MXPA04007585A - Method for producing recombinant proteins in micro-organisms. - Google Patents

Abstract

The invention relates to a method for producing a recombinant functional plasminogen in micro-organisms, and to a method for identifying plasminogen activators. The nucleic acid sequence coding for the functional part of the plasminogen is fused with a nucleic acid molecule coding for at least one signal peptide. The nucleic acid molecule coding for the plasminogen and the nucleic acid molecule coding for the signal peptide are combined with codons for interfaces of proteases which ensure the separation of the signal peptide. The recombinant plasminogen or the corresponding plasmine is suitable for treating wounds which are slow to heal or not healing, by application of the enzyme in an appropriate formulation.

Description

METHOD FOR THE PRODUCTION OF RECOMBINANT PROTEINS IN MICROORGANISMS Technical Field The human fibrinolytic system includes as a central element the plasmin protease (Pm). On the one hand, plasmin is capable of degrading fibrin and on the other hand of activating the matrix of metalloproteinases (MMPs) and growth factors, which in turn are jointly responsible for the degradation of the extracellular matrix and wound healing. BACKGROUND OF THE INVENTION Plasmin originates from its precursor molecule, plasminogen. Up to now, two physiological activators of plasminogen (also referred to as plasminogen activators, PA) are known. These are the plasminogen activator of the tissue type (tissue-type PA, t-PA) and the urokinase-type plasminogen activator (urokinase-like PA, u-PA). In addition, the system is regulated by a set of protease inhibitors, e.g., a2-antiplasmin. The two most important biological properties of plasminogen and plasmin respectively are directly related to the two different activators. The form mediated by the so-called t-PA is responsible for the homeostasis of fibrin, while the form mediated by u-PA is the most prominent in cell migration and tissue remodeling. It can be shown in particular that in the chronic case of mice deficient in u-PA, wound healing does not occur. The same applies to mice, the genes of which were deactivated for plasminogen and t-PA and u-PA respectively. In addition, the life time of the animals was shortened clearly, which is due inter alia, to thrombosis and organ collapse. A review of the plasminogen / plasmin system was published by Desire Collen (Thrombosis and Haemostasis, 82, 1999 (1)). The therapeutic use of plasmin is suitable for the treatment of patients with heart attack or shock, in the case of which a rapid dissolution of the fibrin clot is essential for survival and thus represents an alternative treatment for someone with plasminogen activators, the which achieves the hydrolysis of the fibrin clot only indirectly. The aforementioned mouse models show that plasmin is also a potential therapeutic, which can be used in the treatment of non-wound healing or only slow. Normally, the activation of plasminogen by t-PA occurs only in the presence of fibrin, such as after the termination of the blood coagulation cascade. In the absence of a substrate plasmin is inhibited almost immediately by a2-antiplasmin. This interaction is admittedly slow through the binding of plasmin to fibrin and the degradation of the fibrin clot is activated by this. Different strategies of plasminogen activation are used for therapy, since in the case of an attack or cardiac shock, the dissolution of the blood clot is frequently inevitable for the survival of the patients. For example, infusion of streptokinase leads to rapid recanalization of vessel lumens. Plasminogen activation therapy with streptokinase, a bacterial protein, is not based on a proteolytic activation but on a complex. Then this complex can activate other molecules of plasminogen to plasmin. In addition, a urokinase is used therapeutically, whose admission as streptokinase can not distinguish the fibrin-binding plasminogen from the free plasminogen on a molecular level. Therefore, recombinant human t-PA was developed, which proved itself superior to streptokinase in clinical studies. But these diagnoses were not confirmed by other studies. Precisely the activators of the recombinantly produced plasminogen such as rt-PA (more different derivatives), recombinant single chain urokinase-PA and recombinant staphylokinase that emphasizes the importance of the production systems produced with the genetically molecular methods for the production of recombinant proteins for the use in modern therapy. Plasminogen is the precursor molecule of the plasmin of the fibrinolytic enzyme. The cDNA (Malinowski et al., Biochemistry, 23, 1984 (12); Forsgren et al., FEBS Lett. 213, 1987 (2)) as well as the non-coding introns inclusive gene (Petersen et al., J. Biol. Chem., 265, 1990 (3)) for human plasminogen have already been published in the scientific literature. Human plasminogen (hPg), the plasmin proenzyme of the serine protease, is a glycoprotein consisting of a polypeptide chain of 791 amino acids with a molecular weight of 92,000 and a theoretical isoelectric point of 7.1. The carbohydrate rate is 2% (Collen, 1999, (1)). Plasminogen is produced in the liver, the plasma concentration is approximately 200 mg / 1 (1.5-2 μ?). The molecule is divided into 7 structure domains, -associated with this is the N-terminal preactivation peptide (Glu-1-Lys-77), five partially homologous Kringle domains and the catalytically active proteinase domain (Val-562- Asn-791; Collen, 1999 (1)). The motif of the structure of the common catalytic triple to all serine proteases consists of the amino acids His-603, Asp-646 and Ser-741. The Kringle domain 1 serves as a recognition sequence to bind the plasminogen to fibrin (Petersen et al., 1990 (3)) and different cell surface receptors. Among the post-translational modifications, the two essential glycosylation sites Asn-289 and Thr-346, both located in the Kringle 3 domain, are especially emphasized for the function of the plasminogen (activation capacity through the various proteinases and streptokinase respectively , receptor binding properties). Considering these two forms of main modifications of the plasminogen, the following are distinguished: plasminogen I represents the glycosylation pattern previously described plasminogen II lacks the modification in Asn- 289 Another glycosylation site is the amino acid Ser-248. The amino acid Ser-248 can exist in the phosphorylated form. The activation takes place in the organism through the proteolytic cleavage between the amino acids Arg-561 and Val-562. Subsequently, another proteolytic activation takes place between Lys-77 and Lys-78 towards Lys-78-hPg. Alternatively, this linkage can also be initially hydrolyzed directly into Glu-Pg. The active plasmin Lys-78-hPm binds through the disulfide bridges in each case. Therefore the heavy chain of hPm (1 / 78-561) is responsible for the interaction with substrates, e.g., fibrinogen and fibrin. The light chain (562-791) resulting from the C-terminal represents the catalytically active subunit. Well known in the literature is a method, which was used for the recombinant production of the plasminogen binding domain of fibrin in Pichia pastoris with a yield of 17 mg / 1 (Duman et al., Biotechnol Appl. Biochem. 28; 39-45 1998 (4)). The glycosylation of this domain (ringle 1-4) could be proved by the authors. Another citation describes the production of the two Kringle 4 and 5 domains of human plasminogen (Guan et al., Sheng u Gong Cheng Xue Bao, 17, 2001 (5)). The objective was to identify the domain, which can inhibit the growth of endothelial cells. However, the plasminogen domains recombinantly produced by the two working groups in Pichia pastoris do not possess the decisive catalytic domain for physiological functionality. Gonzalez-Gronow et al., (Biochimica et Biophysica Acta, 1039, 1990 (6)) compared the expression of recombinant human plasminogen in Escherichia coli and COS cells, an simian kidney cell line. Microbial production failed in E. coli, which is attributed by the authors to inadequate glycosylation. The production of the polypeptide chain was successful, but in a form not capable of activation, i.e., the treatment with activators (urokinase and t-PA) did not result in active plasmin. Absent glycosylation results in a protein, which lacks important physiological functions with respect to the activation capacity (undetectable enzyme activity) as well as with respect to endothelial cell recognition (González -Gronow et al., Biochimica et Biophysica Acta, 1039, 1990 (6)). In addition, post-translational modification with carbohydrates significantly influences the half-life in the blood of mammals. It is considered that the authors can produce functional plasminogen in COS cells. Other authors describe functional expression in insect cells (Whitefleet-Smith et al., Arch. Biochem. Biophys., 271, 1989 (7)). However, in the use of mammalian and insect cells the culture conditions are slow and high cost as well as feasible, the low amounts of protein are disadvantageous. Furthermore, in mammalian cells they are not suitable for producing large amounts of a proenzyme due to intracellular expression and to proteases in the cytoplasm (Nilsen and Castellino, Protein Expression and Purification, 16, 1999 (8) and Busby et al., J. Biol. Chem., 266, 1991 (9)). Typically in the baculovirus / lepidoptera system (insect cells) the expression yields are only in the range of 3-10 mg / ml. In WO0250290 the recombinant production of functional mini- and micro-plasminogen in yeast was described. For this, the authors expressed the genes for the catalytic domain of human plasminogen with (mini-plasminogen) or without a Kringle domain (micro-plasminogen) in the host organism Pichia pastoris. The mini- and micro-plasminogen thus produced recombinantly respectively were subsequently purified, processed to mini- and micro-plasmin respectively and their activity was demonstrated in the animal experiment. The claimed production of the recombinant proteins is at 100 mg / 1 by mini-plasminogen and at 3 mg / 1 by micro-plasminogen. However, the higher the protein, the more difficult it is for its recombinant production, which is confirmed in the description of O0250290 by the clear decrease in the production of micro to mini-plasminogen in the order of two decimal exponents. No example of an embodiment for the expression of the larger plasminogen variants such as Lys- or Glu- plasminogen is presented. The recombinant production of functional plasminogen in microorganisms has not been described yet, so that one skilled in the art can execute it. Therefore, it is the aim of the present invention to produce a functional human plasminogen method at low cost and process it into the catalytically active plasmin. This object is solved by a recombinant production method for the production of plasminogen with a microorganism according to claim 1. Additional solutions are mentioned in the independent claims. The dependent claims reflect the preferred embodiments. Surprisingly it was found that recombinant microbial production of Glu-o Lys-functional plasminogen is possible in microorganisms. In addition to this, it was found that the recombinant production of the micro-mini- plasin of Lys and Glu is possible in large quantities not expected. The subject matter of the invention is the cloning of the plasminogen gene into the preferred expression vectors of the micro- and mini- and most preferred plasminogen gene of the plasminogen gene Glu or Lys or in each case of a functional variant thereof and the recombinant production of functional plasminogen, preferably human functional plasminogen using molecular genetic methods.

In addition, the invention describes the identification of proteases, which catalyze the activation of plasminogen towards plasmin. Plasminogen and plasmin respectively, which is produced by this invention, is free of contaminants such as animal or virus proteins, which occur naturally upon isolation from humans, cattle and other mammals and which they can leave side effects in patients. The invention is characterized by a recombinant production method comprising at least the following step: a.) Fusing the nucleic acid sequence encoding at least the functional part of the plasminogen peptide by coupling with a nucleic acid sequence that encoding at least one signal peptide, the nucleic acid sequence encoding the peptide functional plasminogen and the nucleic acid sequence encoding at least the signal peptide with the codons for the cleavage sites of proteases provided for the splitting of the signal peptide. The production of the therapeutic proteins is carried out in an increased manner with the recombinant production systems. Due to the cost factors, we fight to carry out the recombinant production in microbes, especially in bacterial organisms. These systems involve the advantage that along with a comparatively low price production, the productions of the protein can be carried out in the g / 1 range and the recombinant proteins are not contaminated with viruses or proteins such as prions, which can be dangerous for patients. As systems bacterial production are with unable frequency correctly produce the protein several times, the production often takes place in eukaryotic systems such as yeast, insect cells or mammalian cells also reverse in vitro folding of proteins not folded Strains of eukaryotic production and cell production lines offer the advantage that proteins glycosylated with them can be produced. It is especially applied to insect cells or mammalian cells, where the production of the recombinant protein is very expensive and the yields are often very low. They also have the disadvantage that they can also be contaminated with viruses and proteins that are harmful to humans. This is not the case when using eukaryotic microorganisms. Equipment instrumental for growing eukaryotic microorganisms is comparable to that of bacterial microorganisms, contamination with mammalian viruses and proteins are not present and are also possible yields of the protein in the range g / 1. Especially preferred is a eukaryotic host organism that is counted for the yeast section, preferably for the Ascomycota. It is also preferred that it be counted for Saccharomycotina, especially for the class of Saccharomycetes, especially here for the order of Saccharomycetales. According to the especially preferred embodiments, the host organism is also counted for the Saccharomycetaceae family, here especially for the genus Pichia. Preferred eukaryotic microorganisms used according to the invention are emplificativamente eg Saccharomyces cerevisiae for baking, other examples are Candida, the methanotrophic yeast Pichia pastoris, Pichia methanolica and Hansenula plymorpha or filaments fungus genus Aspergillus, such as Aspergillus niger, Aspergillus oryzae and Aspergillus nidulans. Pichia pastoris is especially preferred. The method for recombinant production also characterized that a coding nucleic acid molecule to at least the functional portion of plasminogen is incorporated into an expression vector for this organism, the coding nucleic acid molecule preferably for plasminogen human is fused with the coding of the nucleic acid molecule for at least one signal peptide, preferably a prepropeptide, preferably for transport to the endoplasmic reticulum, the codons for cleavage sites of the proteases that provide the cleavage of the signal sequence or the prepropeptide in the host organism are inserted between the two nucleic acid molecules. Preferably, a nucleic acid molecule encoding human plasminogen is used. In addition, a nucleic acid molecule encoding the nucleic acid molecules of the human plasminogen encoding the plasminogen from other mammals can be used. This allows the production of the plasminogen of the respective mammals. Furthermore, the recombinant human plasminogen is formed according to the present method by overexpression and can, if desired, be secreted into the culture medium from which it can be separated from the host cells through centrifugation, filtration or sedimentation and can be subjected to to the purification of protein without the complex processes of cellular disruption, which can be carried out by methods known to the person skilled in the art. Plasminogen activation in plasmin is resolved by proteases, which are capable of processing plasminogen into catalytically active plasmin. In the following terms used in the context of the present invention are defined: "Method for recombinant production" means, that the peptide or a protein is expressed from a nucleic acid sequence, preferably a DNA sequence through an appropriate host organism, the nucleic acid sequence was formed from a cloning and a fusion of the individual sections of nucleic acid. "Cloning" must hereby include all known cloning methods according to the state of the art, however, which will not be described in detail, because they belong to self-evident tools of the person skilled in the art. "Expression, in a suitable expression system" will comprehend in the present all known expression methods according to the state of the art, especially those, which are mentioned in the claims. Under the "plasminogen-peptide functional part", the part of the plasminogen or plasminogen-peptide will be understood to be able to carry out the biologically relevant functions of the plasminogen. These biologically relevant functions are at least the activation capacity towards plasmin by plasminogen activators such as, for example, tissue plasminogen activator, urokinase, vampiro-bat plasminogen activator, streptokinase, staphylokinase, Pia protein from Yersinia pestis, etc., and proteolytic activity, which is characterized by the hydrolysis of fibrin. The term "plasminogen activator (s)" used in the description and examples will refer to proteolytic as well as non-proteolytic plasminogen activators. Additionally in the case of the plasminogen Glu is understood the processing capacity in the plasminogen Lys through the plasmin-catalyzed cleavage of the preactivation peptide. The ability of increased activation of plasminogen above the factor of 1000 after binding to fibrin, laminin, fibronectin, vitronectin, proteoglycan of heperansulfate, collagen type 4 and other substrates in the same way are counted for biological functions. Among the biologically relevant functions of plasmin, which have to be ensured after plasminogen processing, are the degradation of laminin, the degradation of fibronectin, vitronectin, proteoglycan of heperansulfate, the activation of procollagenase, the activation of promatrix metalloproteases. , activation of latent macrophage elastase, prohormones and growth factors such as TGFP-1 (latent transformation growth factor), VEGF (vascular endothelial growth factor) or bFGF (basic fibroblast growth factor). Another biological function is the ability of inhibition by plasmin inhibitors such as a2-antiplasmin and a2-macroglobulin. The biologically relevant functions are considered in addition to the binding to fibrin, laminin, fibronectin, vitronectin, proteoglycan of heperansulfate and type 4 collagen, binding to receptors such as a-enolase, annexin II or amphotericin. The first of all plasminogens is formed as an inactive Glu plasminogen. This Glu plasminogen can be converted to plasminogen Lys by plasmin through the splitting of the so-called preactivation peptide. Both are converted by tissue plasminogen activators (only in this case through the aforementioned proteolytic activators) through the proteolytic cleavage in plasmin, which consists of subunits connected through the sulfide bridges. The smallest subunit includes the proteolytic domain and the phosphorylation site, the largest subunit carries all three glycosylations and is responsible for binding to fibrin. In addition, glycosylations are important for stability in plasma. Through the formation of a 1: 1 complex with streptokinase and staphylokinase, plasminogen can be further converted into a proteolytically active enzyme, which is capable of processing plasminogen into plasmin. Accordingly, the functional plasminogen is plasminogen that can be processed by the plasminogen activators in the proteolytically active plasmin. In addition, the functional plasminogen preferably includes the fibrin binding domain and may preferably include at least one of the three glycosylations. The smallest forms of functional plasminogen are the micro- and mini-plasminogen, a larger form of Lys plasminogen. The Glu plasminogen, which still includes the preactivation peptide, is also functional plasminogen. However, it is conceivable, that the regions can be omitted especially within the larger chain without significantly interfering with the aforementioned functionalities (inter alia proteolysis, binding to fibrin). It is self-evident for one skilled in the art to produce different forms of plasminogen (referred to as plasminogen derivatives in the following), which include a functional catalytic domain. Under functional, it should be understood, as already described, that the plasminogen varies the characteristics of the proteolytic activity after activation with plasminogen activators such as streptokinase and urokinase. the catalytic domain can comprise amino acid cancellations and exchanges or can be fused with other amino acids or peptides or proteins the large domain can comprise all intermediates from Glu20 to Arg580 (based on the pre-plasminogen sequence), which can be activated with activators of plasminogen in active plasmin As in the precise example, three forms of plasminogen Lys will be mentioned: Variant 1: N-terminal amino acid: Met88 Variant 2: N-terminal amino acid: Lys97 Variant 3: N-terminal amino acid: Val98 Derivatives of plasminogen are approximately preferably a number of 1 to 50 amino acids shorter or larger than the corresponding micro-, mini- Lys or Glu plasminogen or preferably represent an exchange of 1 to 10 amino acids, these derivatives also exhibit the property of being activated by the plasminogen activators. Among the micro-, mini- Lys or Glu plasminogen and corresponding plasminogen derivatives there is sequence homology (sequence equalization) over 80%, preferably over 85%, more preferably over 90%, much more preferred over 95%, it is especially preferred over 98% and more especially over 99% is preferred. Preferably the plasminogen derivatives represent the following characteristics: the catalytic domain can comprise at least one cancellation and / or at least one amino acid exchange and / or be fused with at least one other amino acid or at least one other peptide or at least one other protein. the large domain can comprise all intermediates from Glu20 to Arg580 (based on the pre-plasminogen sequence) that are activated with the plasminogen activators in the active plasmin a plasminogen derivative represents an amino acid sequence homology (equals ) preferred over 80%, more preferred over 85%, even more preferred over 90%, especially preferred over 95%, and more especially preferred over 99%. With "microorganism" are included all forms of life, which are characterized only by minor dimensions. Therefore they will understand eukaryotic as well as prokaryotic microorganisms. Especially bacteria, yeast, fungi and viruses are mentioned. "Nucleic acid" will thus comprise DNA with RNA, both in all imaginable configurations, e.g., in the form of double-stranded nucleic acid, in the form of single-stranded nucleic acid, combinations thereof, as well as linear or circular nucleic acids. "Signal sequence" is understood to mean a sequence of peptides that is capable of ensuring the transport of another sequence of peptides in or through a membrane, e.g., in the endoplasmic reticulum. Therefore, a prepropeptide, a prepeptide or a propeptide can be conceivably envisaged. With "cleavage site" such points are indicated in a sequence of peptides, which are supplied for the unfolding of a signal sequence, a prepropeptide or propeptide from another sequence of peptides or generally the unfolding of a sequence of peptides in two. parts in a host organism. A "nucleic acid coding for at least one peptide or signal prepropeptide" is a nucleic acid sequence, which encodes a peptide or protein structure, which provides the other polypeptide with a transfection in the membranes, eg, in the endoplasmic reticulum. An initiator oligonucleotide is indicated by "primer". In the present it means oligorribo- or single chain, short chain deoxyribonucleotides, which are complementary to a region in a single-stranded nucleic acid molecule and can be hybridized with a double-stranded one. The 3-hydroxy free ends in this double-stranded form serve as a substrate for DNA polymerases and as a starting point for the polymerization reaction of the entire single-stranded double-stranded molecule. The primers are used especially in PCR, i.e., the polymerase chain reaction known to the person skilled in the art. With "plasmid" are indicated nucleic acid molecules, which are not integrated into the chromosome and occur in many prokaryotic and some eukaryotic microorganisms with a length of approximately 2 kb to more than 200 kb. "Ligation" is the term for the connection of the ends of two nucleic acid molecules by means of a ligase or in line with a self-ligation, ie, through an intramolecular ring closure reaction, in which both Single-stranded ends of a linear DNA molecule dimerize as long as their ends can form base pairs with each other. "Restriction endonuclease" is the term for a class of bacterial enzymes, which unfold the phosphodiester linkages with the specific base sequences in both strains of a DNA molecule. "Electroporation" is a method to introduce nucleic acids into cells. Therefore, the cell membranes of the receptor cells, which are located in suspension and grow exponentially, become permeable to the high molecular molecules by brief electrical impulses of high field strength while exposed to the nucleic acid solution. Under "overexpression" is meant an increased production of the functional plasminogen by a cell compared to a production by the wild type of this cell. Normally an overexpression deals with, when the expressed external gene amounts to approximately 1-40% of the total cellular protein of the host cell in case of intracellular production. Under "expression vector" should be understood such vectors, which allow the transcription of the cloned end gene in the vector and the subsequent translation of the mRNA formed (messenger RNA) after being incorporated into a suitable host cell. Expression vectors usually contain the control signals, which are necessary for the expression of genes in prokaryotic and eukaryotic cells. In the promoters of the present invention which are preferably inducible by methanol such as the AOX1 promoter or especially preferred constitutive promoters such as the YPT-1 promoter or the GAP promoter are used for the control of gene expression in yeast such as Pichia pastoris Especially preferred is the constitutive GAP promoter. "A0X1" is a gene of alcohol oxidase 1 from P. pastoris; "GAP" is a glyceraldehyde-3-phosphate dehydrogenase gene from P. Pastoris and "YPT1" is a gene of a GTP-binding protein from P. pastoris. The signal peptides of the proteins encoded by the PHO-1, SUC-2, PHA-E or alpha-F genes are frequently used for secretory production in yeast. "PHOl" is a gene of acid phosphatase from P. pastoris; "SUC-2" is a secretory invertase gene from S. cerevisiae; "PHA-E" is a gene of acid phosphatase from Phaseolus vulgaris Agglutinis; and "alpha-MF" is a gene of alpha mating factors from S. cerevisiae. Especially preferred are the codons for the cleavage sites of the proteases and codons for the cleavage sites for the cleavage of the propeptide for the Kex2 protease or the Stel3 protease. It is especially preferred that the connection takes place in step a) above the codons, which code for a cleavage site Kex2 and additionally two cleavage sites Stel3. In a preferred embodiment of the present invention the nucleic acid molecule encoding the signal peptide or the prepropeptide comes from the yeast, especially from the yeast Saccharomyces cerevisiae. A more preferred embodiment is directed to a nucleic acid molecule encoding the signal peptide or the prepropeptide, which codes for the signal peptide or prepropeptide of the a-factor of the yeast Saccharomyces cerevisiae. The formed fusion product described above in step a) is preferably amplified by PCR and then further in the purified one preferably. In WO02 / 50290 the recombinant production of the mini- and micro-plasminogen is described with the expression vector pPICZocA suitable for the yeast containing the inducible AOXl promoter and the propeptide of the alpha factor yeast. These smaller plasminogen variants have either (such as the micro-plasminogen) absolutely or not or only a Kringle domain (such as mini-plasminogen). The expression vector pPICZaA contains the cleavage sites for the Kex2 and Stel3 proteases. However, Stel3 cleavage sites were canceled in the cloning of the corresponding expression vectors of the mini- and micro-plasminogen. A set of promoters is known for the inducible expression systems in yeast. Included here are the promoter A0X1, A0X2, CUP1 (Koller A, Valesco J, Subramani S., Yeast 2000: 16 (7), 651-6), PHOl (EP0495208), HIS4 (US 4885242), FLD1 ( Shen et al., Gene 1998: 216 (1), 93-10) and the XYL1 promoter (Den Haan and Van Zyl, Ap 1. Microbiol. Biotechnol., 2001: 57 (4), 521-7). By means of the inducible AOX1 promoter of methanol the production of the heterologous protein can be targeted selectively and a homogeneous biomass can be obtained. Before the foreign protein expression is induced, the host organisms can carry out a high growth density without the disadvantages of selection, which can occur in the expression of an alien protein. Contrary to its smaller variants, which are expressed in WO02 / 50290 under the control of the A0X1 promoter, the Glu and Lys plasminogen produced recombinantly in the present invention includes all five Kringle domains, which complicates its recombinant production due to the following reasons: possible loss of the expression cassette due to the growth disadvantages for host organisms in the expression of foreign proteins; - Proteolytic degradation of expressed proteins and low yield The production of Glu- or Lys plasminogen is not described in WO02 / 50290 due to the disadvantages described. These difficulties were solved in the present invention inter alia in the manner that the recombinant protein includes a signal peptide, an ex2 and at least one Stel3, preferably two Stel3 protease cleavage sites. Furthermore, in a preferred embodiment, a feed with glycerol was carried out as another carbon source between 0.1 and 10 ml / h, preferably between 0.5 and 5 ml / h, more preferably between 0.8 and 1.5 ml / h and the culture medium is buffered to a neutral pH of 7.0. Attention was paid for sufficient oxygen feed. In a preferred embodiment, attention was paid to integrate the recombinant nucleic acid without the connection to the 5 'site of the AOX1 gene, but in relation to the 5' site of the glyceraldehyde phosphate dehydrogenase gene from P. pastoris. In this, a non-inducible but a constitutive promoter was used. The constitutive promoters that are activated in yeast and that can be used are the GAP promoter, the YPT1 promoter (Sears et al., Yeast 1998: 14 (8), 783-90), the TKL promoter (Den Haan and Van Zyl, Appl. Microbiol Biotechnol 2001: 57 (4), 521-7), the ACT promoter (Kang et al., Appl Microbiol Biotechnol 2001: 55 (6), 734-41) and the PMA1 promoter (Yeast 2000 : 16 (13), 1191-203). The preferred promoters are the GAP promoter and the YPT1 promoter. A particularly preferred promoter is the GAP promoter. Contrary to an inducible promoter, a constitutive promoter has the disadvantage that the non-expressed protein is produced constitutively during the entire growth phase. Through this, the disadvantages occur for the host cell, which was shown inter alia in slow growth. Because the selection pressure prevails, host cells that have lost the recombinant expression cassette have an advantage and can grow above the recombinant host cells. Through this, a heterogeneous mixed population can arise, which should be avoided. However, it was surprisingly found that the constitutive GAP promoter enables high performance according to a preferred embodiment of the present invention. Although the Lys plasminogen of the AOX1 promoter is used, the yield was obtained after 120 hours of the induction of at least 17 U / 1 (= 1.5 mg / 1), more preferred 120 U / 1 (= 11 mg / 1), more preferred 180 U / 1 (= 16 mg / 1), even more preferred 200 U / 1 (= 18 mg / 1), more preferred 220 U / 1 (= 20 mg / 1), even more preferred 240 U / 1 ( = 22 mg / 1), especially preferred 260 U / 1 (= 24 mg / 1) and more especially preferred 280 U / 1 (= 25.5 mg / 1),, the yields were significantly higher when using a constitutive promoter, especially the GAP promoter. In a preferred embodiment a constitutive promoter, eg, the GAP promoter is operably coupled to a nucleic acid, which codes for at least the functional part of the plasminogen sequence and which is fused to the nucleic acid sequence encoding at least one signal peptide, the sequence of nucleic acids encoding the functional plasminogen and the nucleic acid sequence coding for at least the signal peptide that is coupled to the codons for the cleavage sites of the proteases, which are provided for the splitting of the signal peptide. In a particularly preferred embodiment a constitutive promoter, eg, the GAP promoter, is operably coupled to the plasminogen nucleic acid sequence micro-, mini- Lys or Glu, which is fused to the nucleic acid sequence of a signal peptide at from the yeast. In this regard, it was surprisingly found that the constitutive GAP promoter according to a preferred embodiment of the present invention enables a yield, which is approximately 10 times higher (see example 7c, Lys plasminogen production, 1375 U / 1, which converts the results to 125 mg / 1). In another preferred embodiment, a glycerol feed is carried out as another carbon source between 0.1 and 10 ml / h, preferably between 0.5 and 5 ml / h, more preferably between 0.8 and 1.5 ml / h and the culture medium is buffered at a neutral pH of 7.0. Therefore the growth rate μ [l / h] reaches values between 0.002 and 0.10, preferably between 0.004 and 0.020, more preferably between 0.008 and 0.010. When using the plasminogen Lys of the GAP promoter the yields were obtained after a fermentation time of 250 hours of at least 660 U / 1 (60 mg / 1), preferably 1000 U / 1 (= 91 mg / 1), preferably 1500 U / 1. { = 136 mg / 1), more preferably 2000 U / 1 (= 182 mg / 1), especially 2500 U / 1 (= 227 mg / 1) and more especially 2750 U / 1 (= 250 mg / 1) . They were obtained in the recombinant production according to the plasminogen mini- and higher micro yields. The yields in the case of mini-plasminogen are between 100 mg to 2 g per liter, preferably 300 mg / 1 - 1.5 g / 1, more preferably from 400 mg / 1 - 1 g / 1, additionally more preferably from 500 mg / 1 - 800 mg / 1 and more particularly preferred is 600 - 700 mg / 1. The yields of the micro-plasminogen are also at least 10% higher than those of the mini-plasminogen. The insignificantly lower yields were obtained in the recombinant production of the Glu plasminogen compared to the Lys plasminogen. The method according to the present invention is suitable for the production of mini-, micro-Lys- and Glu plasminogens. Preferred embodiments herein focus on the recombinant production of the mini-plasminogen, micro-, Lys- and Glu-, which are each coupled to a signal or prepro sequence, in an expression vector, containing a constitutive promoter, eg, the GAP promoter. In a preferred additional embodiment, the signal sequence consists of the signal peptide or prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae. In a particularly preferred embodiment a constitutive promoter, e.g., the GAP promoter, is operably coupled to a nucleic acid of the Sec sequences. ID. No. 7 or 9 or one of the sequences Sec. ID. No. 13 or 15 or one of the sequences Sec. ID. No. 50 to 59 and is expressed in an appropriate expression vector. In a further preferred embodiment a constitutive promoter, e.g. the GAP promoter is operably coupled to a nucleic acid, which codes for at least the functional part of the plasminogen sequence. In a particularly preferred embodiment a constitutive promoter, e.g., the GAP promoter is operably coupled to a nucleic acid of the Sequences ID. No. 13, 15, 7 and 9 or one of the sequences Sec. ID. No. 50 to 59 or Sequence Sec. ID. No. 11 and is expressed in an appropriate expression vector.

Plasminogen Glu (data calculated with the EditSeq ™ program (DNASTAR)) Molecular weight: 88431.67 Daltons 791 amino acids isoelectric point: 7,121 charge at pH 7.0: 1,351 Glycosylation sites: 0-268, N-308, 0-365 (the numerals are refer to pre-plasminogen consisting of 810 amino acids) Plasminogen Lys (data calculated with the program EditSeq ™ (DNASTAR) Molecular weight: 79655.71 Daltons 741 amino acids isoelectric point: 7,492 charge at pH 7.0: 5,287 Glycosylation sites: 0-268, N-308, 0-365 (the numerals refer to pre-plasminogen consisting of 810 amino acids) Mini-plasminogen (data calculated with the EditSeq ™ program (DNASTAR) Molecular weight: 38169.63 Daltons 348 amino acids isoelectric point: 7,203 charge at pH 7.0: 0.893 Glycosylation sites: none Micro-Plasminogen (data calculated with the EditSeq ™ program ( DNASTAR) Molecular weight: 27230.41 Daltons 249 amino acids isoelectric point 7.934 at pH 7.0: 3.733 Glycosylation sites: none The method according to the invention is described in detail below The fusion product generated in step a) of the present invention it can be further implemented in a suitable expression vector for microorganisms.This expression vector is preferably chosen from the group comprising pPICZaA, B and C and pPICZA, B and C and pGAPZaA, B and C and pGAPZA, B and C and pPIC6aA, B and C and pPIC6A, B and C as well as pA0815, pPIC3.5K and pPIC9K. The introduction into the expression vector is preferably carried out again by ligation. The PCR product as well as the expression vector are preferably cut with the restriction endonucleases Kspl and Xhol, before they are ligated with a T4 DNA ligase. The ligated nucleic acid can be transformed through electroporation into a microorganism, preferably E. coli and the DNA can be isolated from the transformed strains obtained in that form and separated through endonucleolytic cleavage preferentially with Xhol or Sful and Kspl. The nucleic acid obtained in that form can be a plasmid preferably chosen from the group pMHS476.1, pSM54.2, pSM49.8, pS 82.1, and pSM58.1, pAC37.1, pJW9.1, pPLGl.l, pPLG2 .1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1, pPLGIO.l, pPLG11.2, pPLG12.1, pPLG13.1, pPLG14.2 , pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 and pPLG20.1. As an initiator for the aforementioned amplification, two oligonucleotide primers preferably chosen from the group comprising N034 (sequence ID-No.1), N036 (sequence ID- No. 2), N036a (sequence ID-No • 19) are used. , N036b (sequence ID -No ), N036c (sequence ID-No.21), N036d (sequence ID-No. 22), N036e (sequence ID-No.23), N036f (sequence ID -No- 24), N036g (sequence ID-No.25), N036h (sequence ID -No 26), N036Í (sequence ID-No.27), N036j (sequence ID -No 28), N057 (sequence ID-No.3), N037 (sequence ID-No.4) N035 (sequence ID-No.5) and N056 (sequence ID-No.6). According to the present invention, the following modalities are especially preferred: The codons encoding the cleavage site of the Kex2 protease and the plasminogen fusion gene, which represent the nucleic acid sequence shown in sequence ID No. 7 or 13. The codons coding for the cleavage site of the Kex2 protease and the plasminogen fusion protein, which represent the nucleic acid sequence shown in sequence ID No. 8 or 14. The codons coding for the site of cleavage of the Kex2 protease and the protease Stel3 and the plasminogen fusion gene, which represent the nucleic acid sequence shown in sequence ID No. 9 or 15. The codons coding for the Kex2 protease and the Stel3 protease and the plasminogen fusion protein, which represent the nucleic acid sequence shown in sequence ID No. 10 or 16. Preferably the plasmid above me ncionado, which is preferably chosen from the above mentioned group, is transformed into a microbial host. The transformation can be carried out, for example, by electroporation. The microorganism used is preferably a eukaryotic microorganism that is counted for the section of the fungus. Preferred microorganisms are counted for Ascomycota, Saccharomycotina is preferred and hence the Saccharomycetes class is preferred, the order of Saccharomycetales is further preferred, more preferably the Saccharomycetaceae family and hence the Pichia, Saccharomyces genus is especially preferred. , Hansenula. and Aspergillus. According to a particularly preferred embodiment of the present invention, the nucleic acid sequence encoding at least the functional part of the plasminogen is overexpressed from a microbial host organism transformed with the fusion product generated in step a) described above and at least the functional part of the plasminogen is secreted, preferably it is secreted in the culture medium. According to another preferred embodiment, the functional part of the plasminogen nucleic acid sequence is one of the ID-No sequences. 60, 61, 62, 63, 64, 65 or 66. According to another preferred embodiment, the functional part of the plasminogen nucleic acid sequence corresponds to the complete plasminogen sequence. Preferably, a human functional plasminogen is produced with the method of recombinant production according to the present invention. This plasminogen, which can be obtained by the method of recombinant production according to the present invention or of the plasmin that results from the influence of the proteases thereof, can be used for the production of a pharmaceutical for the treatment of wounds, especially in the treatment of wounds that heal poorly or slowly, for the treatment of thrombotic events or for the prevention of thrombotic events.

Furthermore, it was detected that the plasminogen produced according to the present invention as well as the plasmin obtained therefrom represent the anticoagulant properties. These advantageous properties allow the addition of the use of plasminogen and / or plasmin as an anti-thrombotic active agent as well as anti-coagulant for the prophylaxis and / or treatment of heart attack, thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammations of the blood vessels, as well as for the treatment subsequent to a cardiac attack, subsequent to the surgical revascularization, subsequent to an angioplasty as well as to a distention of the cavity. Plasminogen can also be used for thrombotic therapy in the case of acute heart attack, for recanalization of arteriovenous shunts as well as for reperfusion of the occluded coronary arteries in the case of an acute heart attack. Additional uses of the plasminogen produced according to the invention include the prophylaxis and treatment of acute pulmonary embolism, of recent coagulations or past venous thromboses, acute or subacute arterial thrombosis, venous thrombosis, acute arterial occlusions of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep venous thrombosis of the hip and extremities, early thrombosis in the area of de-blistered vessels, acute occlusion of the central vessel in the eye, conjunctivitis in the case of type-1 deficiency of plasminogen, burns and freezing injuries , burns by acids or alkalis as well as disseminated intravasal coagulation during the attack. In the case of these indications plasminogen and / or plasmin are preferably used together with an anticoagulant. Heparin, heparin derivatives or acetylsalicylic acid are suitable as anticoagulants. The present invention therefore also focuses on pharmaceutical compositions comprising a plasminogen, which has been produced according to the method of the recombinant production of the present invention, or the plasmin obtained therefrom, in combination with a substrate, additive and / or pharmaceutically acceptable solvent when required. In addition, the pharmaceutical compositions may preferably contain an anticoagulant active agent, especially heparin, heparin derivatives or acetylsalicylic acid. The plasminogen produced according to the invention and / or the plasmin obtainable therefrom are used in the external treatment of wounds preferably in pharmaceutical compositions which are suitable for topical application. By this the plasminogen and / or the plasmin are used in a concentration of 0.01-500 U per gram of pharmaceutical composition, 0.1-500 U is preferred, 0.1-250 U is further preferred, more preferably 0.5-250 U per gram of Pharmaceutical composition and is especially preferred at a concentration of 1-150 U of plasminogen and / or plasmin per gram of pharmaceutical composition. If the plasters or other materials for restoration are used in place of semi-solid formulations in the form of for example ointments, pastes, gels, etc. , the regions of concentration previously given by 2 cm2 of the poultice surface and surface of the materials to be restored respectively are considered. The pharmaceutical compositions according to the invention are produced with the common solid or fluid substrates or diluents and the pharmaceutical auxiliary agents commonly used according to the desired type of application in a suitable dosage in a known manner. Preferred formulations or pharmaceutical compositions are present in a pharmaceutical form, which is suitable for local external application. Such pharmaceutical forms are for example ointments, pastes, gels, coatings, dispersions, emulsions, suspensions or special formulations, such as nanodisperse systems in the form of liposomes., nanoemulsions or lipid nanoparticles, as well as free surfaide formulations, stabilized polymer emulsions or stabilized particle emulsions. The methods for the production of various formulations as well as the different methods of application are known to the person skilled in the art and are described in detail for example in "Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton PA". The compositions produced by parenteral application are suitable in the case of using the pharmaceutical compositions for the prophylaxis and / or treatment of heart attack, thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, inflammations of blood vessels, acute heart attack as well as as for the subsequent treatment of the cardiac attack, subsequent to a revascularization by surgery, subsequent to an angioplasty as well as subsequent to a distention of the cavity. Furthermore, the pharmaceutical compositions are suitable in the case of various systemic applications comprising the use in the case of acute lung embolism, thrombotic therapy in the case of acute heart attack, recent coagulations or past venous thrombosis, acute or subacute arterial thrombosis, recanalization. of arteriovenous shunts, venous thrombosis, reperfusion of occluded coronary arteries in the case of acute heart attack, acute arterial occlusions of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep vein thrombosis of the hip and extremities, early thrombosis in the area of deobliterated vessels, acute occlusion of the central vessel in the eye, conjunctivitis in the case of type 1 deficiency of plasminogen, burn injuries, alkaline or acidic burns and freezing, disseminated intravasal coagulation during the attack. Another possibility for the application arises in the case of plasminogen deficiencies, such as hereditary or congenital plasminogen deficiency (homozygote type 1 plasminogen deficiency), which may result e.g., in lignose conjunctivitis or thrombophilia. There is a possibility here of treating the disease through for example the intravenous administration of the recombinant plasminogen, including in the Glu-, Lys-, mini- and micro-plasminogen forms as well as the variants derived therefrom (Heinz efc al ., Klin, Monatsblatt Augenheilkunde 2002, 219 (3): 156-8). In another possibility for the application, the resolution of the pseudomembranes and the normalization of the respiratory passages can be achieved as well as the improved wound healing in the case of the administration of the plasminogen. This application was described for a newborn child (The New England Journal of Medicine 1998, 339, 23, 1679-1686). Thus, the recombinantly produced plasminogen is potentially used together with the plasmin obtained therefrom or also only plasmin in pharmaceutical compositions, which is suitable for the prophylaxis and / or treatment of acute pulmonary embolism, thrombotic therapy in the case of acute cardiac attack, recent coagulations or past venous thrombosis, acute and subacute arterial thrombosis, recanalization of arteriovenous shunts, venous thrombosis, reperfusion of occluded coronary arteries in the case of acute heart attack, acute arterial occlusions of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts, deep venous thrombosis of the hip and extremities, early thrombosis in the area of de-blistered vessels, acute occlusion of the central vessel in the eye, conjunctivitis in the case of type-1 deficiency of plasminogen, burn injuries, burns alkaline or acidic and freezing, coag intravasal uration disseminated during the attack. The plasminogen recombinantly produced according to the invention is preferably used in pharmaceutical compositions, which are suitable for the topical treatment of burn injuries., frostbite, alkaline or acidic burns, injuries and / or wounds, especially wounds that are poorly healed. Hence, the recombinant plasminogen is preferably used together with at least one activator (plasminogen activators such as, for example, urokinase or streptokinase). Another preferred possibility is to convert the plasminogen produced according to the invention totally or partially before its use via a plasmin activator and to use it in the indications and formulations described herein in the form of plasmin or plasmin with plasminogen. Parenteral applications are especially considered intravenous, intravasal, intraperitoneal, subcutaneous as well as intramuscular application. In the case of parenteral formulations, especially in the form of solutions for injection or infusion, the protein is used in a concentration of 0.1-100 million units, preferably from 1 to 10 million units per 10 ml of solution and especially preferably of 3. to 5 million units per 10 ml of solution. In the case of formulations suitable for oral application, the protein is used in a concentration of 0.1 to 100,000 units per gram of the formulation and especially preferably 1,000 to 50,000 units per gram of the formulation. Additional advantageous formulations are represented, for example, by bandages, dressings or other materials for protease-containing dressings. These formulations are especially suitable for topical application in case of wound healing, or for the treatment of burn injuries, surface freezing, burns and / or alkali or acid lesions. The recombinantly produced plasminogen according to the invention is preferably used in pharmaceutical compositions, especially in wound healing agents, in bandages as well as in dressing materials together with at least one activator (plasminogen activators such as, for example, urokinase or streptokinase) or is converted in advance to plasmin via the activators described above and is used as a plasmin potentially together with the plasminogen and potentially with at least one activator and / or in the pharmaceutical compositions and formulations. Especially preferred is the use of the plasminogen, preferably the plasminogen with an activator, or plasmin, or plasmin together with plasminogen and an activator in and / or on bandages and dressing materials, which are suitable for wound healing, especially for the treatment of poor healing wounds, as well as for the treatment of burn injuries, superficial freezing, burns or other alkali or acidic injuries. The materials for dressings, dressings for wound healing or bandages contain the plasminogen produced according to the invention and / or the plasmin obtained therefrom in a concentration of 0.01-500 units of plasminogen and / or plasmin per cm.2 of the formulation pharmaceutical, preferably from 0.1 to 500 units of plasminogen and / or plasmin per cm.2 of the material for dressings and bandages respectively. Preferably the plasminogen or plasmin is contained in a concentration of 0.1-250 units, even more preferably 0.5-250 units and especially preferably of 1-150 units of the plasminogen and / or a plasmin resulting therefrom by cm.2 of the pharmaceutical formulation in the bandage or apposite material. For the activation of 1 μg of the plasminogen between 100 ^ g and 1 ng of urokinase are used, preferably between 10 μg and 10 ng of urokinase are used. For the activation of 1 μg of plasminogen between 1 mg and 1 ^ g of streptokinase is used, preferably between 300 μg and 3 fig streptokinase are used. For the activation of 1 mg of plasminogen protease of S. griseus between 100 μg and 10 ng are used, preferably between 10 g and 100 ng of protease of S. griseus are used. For the activation of 1 mg of plasminogen between 100 g and 10 ng of protease VIII is used, preferably between 10 g and 100 ng of protease VIII is used. Preferably the nucleic acid sequence encoding the functional part of the plasminogen is a DNA sequence. The present invention further relates to the following plasmids: Plasmid pPLGl .1 Plasmid pPLG2.1 Plasmid pPLG3.2 Plasmid pPLG .2 Plasmid pPLG5.3 Plasmid pPLG6.1 Plasmid pPLG7.1 Plasmid pPLG8.3 Plasmid pPLG9.1 Plasmid pPLGIO. l Plasmid pPLG11.2 Plasmid pPLG12.1 Plasmid pPLG13.1 Plasmid pPLG14.2 Plasmid pPLG15.1 Plasmid pPLG16.3 Plasmid pPLG17.2 Plasmid pPLG18.1 Plasmid pPLG19.2 Plasmid pPLG20.1 Plasmid pMHS476.1 (shell No .: DSM 14678) Plasmid pSM54.2 (Deposit No: DSM 14682) Plasmid pSM49.8 (Deposit No .: DSM 14681) Plasmid pSM82.1 (Deposit No .: DSM 14679) Plasmid pSM58.1 (Deposit No .: DSM 14680 ) Plasmid pAC37.1 (reservoir No .: DSM 15369) Plasmid pJ 9.1 (reservoir No .: DSM 15368). (The deposit numbers refer to the deposit in the German Collection of Microorganisms and Cell Cultures Ltd., Mascheroder Weg Ib, D-38124 Braunschweig). In addition, the present invention relates to a DNA sequence suitable for expression, comprising the nucleic acid sequence encoding at least the functional part of the plasminogen, obtainable by the recombinant production method according to the present invention. It also refers to the host microbial organism, which comprises the fusion product contained in step a) described above and a nucleic acid sequence derived therefrom. Additionally, the present invention relates to a vector, a DNA molecule or an RNA molecule, comprising the fusion product contained in step a) described above or a nucleic acid sequence derived therefrom. Finally, the present invention also relates to a detection method for the identification of plasminogen activators, especially plasminogen activating proteases, while the functional plasminogen is used, produced according to the recombinant production method described above. For this purpose, preferably after the pre-incubation of the proteases the activity of the resulting plasmin is measured with the functional plasminogen produced according to the present invention. The activity of the resulting plasmin can be measured with a synthetic substrate of the peptide. Especially preferably the activity of the resulting plasmin is measured with N-tosyl-Gly-Pro-Lys-pNA. The invention is explained in more detail by the drawings, which illustrate the following: Figure 1: Physical map of plasmid pMHS476.1 (5682 bp). The alpha factor prepropeptide gene is connected through the codons for a Kex2 cleavage site with the human Lys plasminogen gene and is under the control of the A0X1 promoter. Figure 2: Physical map of plasmid pSM54.2 (5694 bp.). The alpha factor prepropeptide gene is connected via the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human Lys plasminogen gene and is under the control of the A0X1 promoter. Figure 3: Physical map of plasmid pSM49.8 (5715 bp). The human preplasminogen gene is under the control of the AOX1 promoter.

Figure 4: Physical map of plasmid pSM82.1 (5913 bp.). The alpha factor prepropeptide gene is connected via the codons for a Kex2 cleavage site with the human Lys plasminogen gene and is under the control of the A0X1 promoter. Figure 5: Physical map of plasmid pSM58.1 (5925 bp.). The alpha factor prepropeptide gene is connected via the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human Glu plasminogen gene and is under the control of the A0X1 promoter. Figure 6: Physical map of plasmid pAC37.1 (11400 bp.). The alpha factor prepropeptide gene is connected via the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human Lys plasminogen gene and is under the control of the A0X1 promoter. Figure 7: Physical map of plasmid pJW9.1 (5925 bp.). The alpha factor prepropeptide gene is connected via the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human Lys plasminogen gene and is under the control of the GAP promoter. Figure 8: Physical map of plasmid pPLGl .1. The alpha factor prepropeptide gene is connected via the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human mini-plasminogen gene and is under the control of the A0X1 promoter. Figure 9; Physical map of plasmid pPLG11.2. The alpha factor prepropeptide gene is connected via the codons for a Kex2 cleavage site and two Stel3 cleavage sites with the human mini-plasminogen gene and is under the control of the GAP promoter. Figure 10: Detection of fibrinolysis activity in the Klárhof test (cleared area). According to the invention, all microorganisms can be considered as host organisms, which are capable of carrying out glycosylation and, if desired, the secretion of proteins. The following are mentioned here: S. cerevisiae, P.pastoris,. P metha.no! Ica and H. polymorpha or the filamentous fungus Aspergillus sp. Especially considered is the use of the functional plasminogen and the plasmin respectively produced according to the present production method in a pharmaceutical composition. In such a formulation the functional plasminogen can be mixed with a pharmaceutically acceptable substrate or auxiliary agent as well as with other suitable auxiliary agents or additives in a manner known to the person skilled in the art. The cleavage site Kex2 provides the cleavage of the propeptide by means of the Kex2 protease located in the Golgi apparatus. This protease also referred to as a YscF protease or as Kexin is a proprotein processing serine protease, which is cut C-terminally from basic amino acid pairs (e.g.: Lys-Arg). The cleavage site Stel3 provides the cleavage of the propeptide by means of the Stel3 protease located in the Golgi apparatus. Stel3 (also referred to as protease YscVI or as dipeptidyl aminopeptidase A) is located in the last Golgi and removes step by step the N-terminal Xaa-Ala dipeptides, e.g., from the immature factor-a of yeast S. cerevisiae. In addition to the cleavage sites for the Kex2 and Stel3 proteases, other cleavage sites can be inserted, which are recognized as substrates by the proteases located in the endoplasmic reticulum or in the Golgi apparatus. It is also possible to fuse with the plasminogen gene exclusively a signal sequence (pre-peptide) responsible for the transport in the endoplasmic reticulum, i.e., the e.g. propeptide, of the pairing factor of the yeast S. cerevisiae is not necessarily required. The microbiological, molecular biological and chemical methods of proteins mentioned in the examples are well known to the person skilled in the art. The following reference books will be mentioned as reference: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989 (10); Gassen & Schrimpf, Gentechnische Methoden, Spektrum Akademischer Verlag, Heidelberg, 1999 (11); EasySelect ™ Pichia Expression Kit Manual Instruction, Invitrogen, Groningen, The Netherlands, Catalog No. K1740-01. Pichia pastoris species and expression systems also come from Invitrogen and are described in the aforementioned Instruction Manual. In the case of pPICZA, B and C in Pichia pastoris short of 3.3 kb, the expression vectors are involved. The vectors have a Zeocin resistance gene for direct selection of Pichia transformants. In addition, the vectors have a C-terminal marker sequence, which provides rapid purification and detection of the fusion proteins. In the case of pPICZalpha A, B and C, the 3.6 kb Pichia pastoris expression vectors are involved, which also have the zeocin resistance gene as well as the aforementioned C-terminal marker sequence. In addition, they contain the secretion signal of the alpha factor of Saccharomyces cerevisiae for an efficient transport of the proteins in the medium. In addition, plasminogen can be activated. In addition, the plasminogen can be exemplary incubated with a protease, which was identified with the detection method according to the invention. Preferably the plasminogen is further incubated with S. griseus protease, protease VIII or protease XVIII, with ficin, metalloendopeptidase, clostripain, with endoproteinase Glu-C, protease XIII, proteinaza A, trypsin, endoproteinase Asp-N or elastase. It is also conceivable to activate the plasminogen by means of the incubation of the plasminogen with one of the proteases t-PA, u-PA or vb-PA (vampire bat PA). In another preferred embodiment, plasminogen is activated by incubation with staphylokinase or streptokinase. Streptokinase or staphylokinase form a 1: 1 complex with plasminogen. By this complex formation, the plasminogen bound in the complex receives a change in conformation, so that it becomes proteolytically active and is able to activate the plasminogen in plasmin. Functional plasminogen or activated functional plasminogen produced according to the present recombinant production method is capable of hydrolyzing fibrin. It is also capable of activating promatriz metalloproteasas and growth factors. The invention will be explained in more detail by the examples as follows. Example: Amplification of the Lys plasminogen gene with insertion of the codons for a Kex2 cleavage site at the 5 'end Plasmid pPLGKG (Forsgren et al., FEBS Lett., 1987 Mar 23; 213 (2): 254-60 ( 2)), which contains the gene for the Glu pre-plasminogen, was isolated from the E. coli HB101 species (pPLGKG) using the QIAGEN plasmid midi kit (QIAGEN, Hilden). 150 ng of pPLGKG-DNA were linearized with 10 U of the restriction endonuclease EcoRI (Roche, Mannheim) and then purified with the Qiaquick PCR purification kit (QIAGEN, Hilden). For the amplification of the plasminogen gene, the pair of oligonucleotide primers N034 (Seq ID No. 1) and N036 (Seq ID No. 2) was used. The oligonucleotide primer N036 has, in addition to the bases complementary to the plasminogen gene, the codons for the cleavage site Kex2. For PCR, 0.5 U of Pwo-DNA polymerase (Hybaid, Heidelberg), each with 400 nM of the oligonucleotide primer, each with 200 μ? Were used. of dNTP, 3 ng of linearized pPLGKG-DNA and the respective reaction buffer in a final volume of 50 μ? . The initiator link temperature was 50 ° C.

The resulting PCR product was tested for the expected size by agarose gel electrophoresis and purified with the QIAquick PCR purification kit. Example Ib: Cloning of the plasminogen gene in the pPICZaA vector 400 ng of the PCR product were cut with every 10 U of the restriction endonucleases Kspl and Xhol (Roche, Mannheim). 300 ng of plasmid pPICZaA DNA (Invitrogen, Groningen, The Netherlands), containing the pre-propeptide sequence of S. cerevisiae factor a, were also cut with 10 U of the restriction endonucleases εpl and Xhol. The DNA thus treated was electrophoretically separated on a 0.9% agarose gel and the fragments obtained were extracted from the gel with the QIAquick gel extraction equipment (QIAGEN, Hildeb). The vector DNA was combined with the insert DNA and ligated at 4 ° C overnight with 1 U of T4-DNA ligase (Roche, Mannheim). The DNA of the ligation batch was then purified with the QIAquick PCR purification kit and used for the transformation of E. coli JM109 by electroporation. The electroporated cells of E. coli JM109 were incubated for lh at 37 ° C in 1 ml of SOC medium, after which they were plated on a solid medium of LB agar with 20 ^ g / μ? of zeocin (Invitrogen, Groningen, The Netherlands) and incubated at 37 ° C overnight.

From one of the E. coli species thus obtained, the DNA was isolated with the mini QIAGEN plasmid kit (QIAGEN, Hilden) and after endonucleolytic cleavage with Xhol and Kspl enzymes, 300 ng were separated by agarose gel electrophoresis. The isolated plasmid contained a fragment of the expected size and was referred to as pMHS476.1 (Figure 1). The correct sequence of the fusion gene from the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae and the Lys-plasminogen gene as well as the codons for the cleavage site sequence of the Kex2 protease were confirmed by sequence analysis (Seq. ID No. 7). Example: Transformation of Pichia pastoris with the plasmid pMHS476.1 Plasmid DNA of the expression vector pMHS476.1 was isolated from the E. coli JM109 species (pMHS476.1) with the plasmid QIAGEN plasmid DNA plasmid. . 10 μg of pMHS476.1-DNA were linearized with 100 U of Pmel (New England Biolabs., Frankfurt) and used for the electroporation of Pichia pastoris KM71H his 4 :: HIS 4 arg 4 aoxl:: genotype ARG 4 of Pichia pastoris Y-11430 (Northern Regional Research Laboratories, Peoria, USA) according to the protocol shown in the Pichia EasySelect ™ Expression Team Instruction Manual. Colonies cultured with 100 μg / ml zeocin after three or four days on YPDS solid medium (Pichia EasySelect ™ Expression Equipment Instruction Manual) were plated on 100 μ? /? of zeocins on YPDS solid medium and were used for the inoculation of liquid cultures. The colonies were referred to as Pichia pastoris KM71H / pMHS476.1-1 / a, while "a" represents the consecutive numbering of the colonies that start at 1. Example Id: Growth of Pichia pastoris KM71H / pMHS476.1-1 / 1 to -1/3 and Induction of Plasminogen Gene Expression For the production of precultures, 100 ml of the BMGY medium was incubated (Pichia EasySelect ™ Expression Equipment Instruction Manual) in containers deflectors from 1 1 to 28 ° C and at 250 rpm up to OD60o = 20-30. After this the precultures were centrifuged for 10 min at 4645 g and at 4 ° C. The cells thus harvested were resuspended in a BMMY medium (0.5% methanol), in order to obtain a bio-wet mass concentration of 80 g / 1. 60 ml of these main cultures were incubated for 118 h in baffles of 300 ml at 28 ° C and 250 rpm. After 24 and 72 hours 2% methanol was added. Baffle containers and high revolutions per minute of 250 rpm were used to provide sufficient oxygen feed, necessary for the use of the AOX promoter. Example: Measurement of plasminogen activity in the supernatant of the main cultures after activation with streptokinase Samples from the main cultures were centrifuged for 10 min at 16,000 g. 300 μ? of the supernatant were incubated for 20 min at 37 ° C with 1 μ? of streptokinase (S8026) (Sigma, Deisenhofen). At 750 μ? 100 mM sodium phosphate buffer, pH 8, 0.36 mM CaCl2, 0.9% NaCl were pipetted 100 μ? of N-tosyl-Gly-Pro-Lys-pNA solution (9.5 mg dissolved in 75 mg of glycine / 10 ml, 2% Tween® 20) and incubated for 10 min at 37 ° C. To start the reaction, 250 μ? of supernatant pretreated with streptokinase and further incubated at 37 ° C. The increase in extinction was measured photometrically at 405 nm. For the determination of the control values, supernatants from a parallel cultivated culture of P. pastoris KM71H were used as well as the supernatants without streptokinase activation. For the samples taken after 72 h of induction, the following activity values were determined (1 U / 1 = 1 μt N-tosyl-Gly-Pro-Lys-pNA conversion per minute per liter of the culture supernatant): KM71H / pMHS476.1-1 / 1: 2 U / 1; KM71H / pMHS476.1-1 / 2: 2 U / 1; KM71H / pMHS476.1-1 / 3: 1 U / 1. After 118 h of induction, the following activity values could be determined: KM71H / pMHS476.1-1 / 1: 7 U / 1; KM71H / pMHS476.1- 1/2: 9 U / 1; KM71H / pMHS476.1-1 / 3: 8 U / 1.

Example 2a: Amplification of the Lys plasminogen gene with insertion of the codons of a Kex2 cleavage site and of two cleavage sites Stel3 at the 5 'end The amplification of the plasminogen Lys gene for cloning within the vector pPICZ A with the insert of the codons for a cleavage site Kex2 and two cleavage sites Stel3 was carried out with the two oligonucleotide primers N034 and N057 (Seq ID No. 3) using the conditions mentioned in example la. The oligonucleotide primer 57 has, in addition to the bases complementary to the plasminogen gene, the codons for the cleavage site Kex2 and the cleavage sites Stel3. Example 2b: Cloning of the Lys plasminogen gene amplified as described in example 2a into the pPICZaA vector The cloning of the plasminogen Lys gene into the pPICZaA vector for the production of a fusion gene of the yeast alpha factor propeptide gene of S. cerevisiae and the plasminogen gene with the insertion of the codons for the cleavage sites of the Kex2 and Stel3 proteases was carried out analogous to the cloning described in example Ib. The obtained plasmid was referred to as pSM54.2 (Figure 2). The correct sequence (Seq ID No. 9) was confirmed by sequence analysis.

Example 2c: Transformation of Pichia pastoris with the plasmid pSM54.2 As described for pMHS476.1 in the example le, Pichia pastoris KM71H was transformed with the plasmid pSM54.2. The obtained colonies were referred to as Pichia pastoris KM71H / pSM54.2-1 / a, while "a" represents again the consecutive numbering of the colonies beginning at 1. Example 2d: Pichia pastoris culture KM71H / pSM54.2- 1/1 to -1/3 and induction of the plasminogen gene The production of the precultures and the main cultures as well as the induction with methanol were carried out analogous to the conditions described in example Id. Example 2e: Measurement of the Plasminogen activity in the main culture samples after activation with streptokinase The activity of the plasminogen after activation with streptokinase was determined as described for K 71H / pMHS476.1-1 / 1 to -1/3 in the example you. For the samples taken after 72 h of induction, the following activity values were obtained: KM71H / pSM54.2-l / l: 2 U / 1; KM71H / pSM54.2-1 / 2: 8 U / 1; K 71H / pSM54.2 - 1/3: 6 U / 1. After 118 h of induction, the following activity values were determined: KM71H / pSM5.2 - l / l: 8 U / 1; KM71H / pSM54.2 -1/2: 17 U / 1; K 71H / pSM54.2-1 / 3: 13 U / 1. Example 3a: Amplification of the plasminogen gene with its own signal sequence and cloning in the pPICZA vector; transformation of Pichia pastoris The amplification of the plasminogen gene inclusive of the sequence coding for the own signal peptide (pre-plasminogen) for cloning in the vector pPICZA was carried out with the two oligonucleotide primers N034 and N037 (Seq ID No. 4) using the conditions described in example la. The cloning of the preplasminogen gene in the vector pPICZA was carried out analogous to the cloning described in Example Ib, while the vector as well as the PC product were cut with the restriction endonucleases Sful and Kspl. The obtained plasmid was referred to as pSM49.8 (Figure 3). The correct sequence (Seq ID No. 11) was confirmed by sequence analysis. As described for pMHS476.1 in the example le Pichia pastoris K 71H was transformed with the plasmid pSM49.8. The colonies obtained were referred to as Pichia pastoris KM71H / pSM49.8-l / a, while "a" again represents the consecutive numbering of the colonies beginning with 1.

Example 4a: Amplification of the plasminogen Glu gene with insertion of the codons of a cleavage site ex2 and cloning into the expression vector pPICZct (alpha) A; transformation of Pichia pastoris Amplification of the plasminogen gene Glu for cloning into the pPICZoA vector with insertion of the codons for a cleavage site Kex2 was carried out with the two oligonucleotide primers N034 and N035 (Seq ID No. 5) using the conditions described in Example la. The oligonucleotide primer N035 has, in addition to the bases complementary to the plasminogen Glu gene, the codons for the cleavage site Kex2. The cloning of the gene plasminogen Glu in the vector pPICZaA for the production of a fusion gene from the gene of the prepropeptide of the alpha factor of the yeast S. cerevisiae and the plasminogen gene Glu with the insertion of the codons for the sites of unfolding of the Kex2 protease was carried out analogous to the cloning described in Example Ib. The plasmid obtained was referred to as pSM82.1 (Figure 4). The correct sequence (Seq ID No. 13) was confirmed by sequence analysis. As described for pMHS476.1 in the example le Pichia pastoris KM71H was transformed with the plasmid pSM82.1. The colonies obtained were referred to as Pichia pastoris KM71H / pSM82. l / a, while "a" represents again the consecutive numbering of the colonies starting at 1. Example 5a: Amplification of the plasminogen gene Glu with insertion of the codons of a cleavage site Kex2 and of two cleavage sites Stel3 at the 5 'end and cloning in the expression vector pPICZotA; transformation of Pichia pastoris The amplification of the plasminogen gene Glu for cloning in the vector pPICZotA with insertion of the codons for a cleavage site Kex2 and two cleavage sites Stel3 was carried out with the two oligonucleotide primers N034 and N056 ( Seq ID No. 6) using the conditions described in example la. The oligonucleotide primer N056 has, in addition to the bases complementary to the plasminogen Glu gene, the codons for the cleavage site Kex2 and the cleavage sites Stel3. The cloning of the plasminogen gene Glu in the vector pPlCZaA for the production of a fusion gene from the gene of the prepropeptide of the yeast alpha factor S. cerevisiae and the plasminogen Glu gene with the insertion of the codons for the sites of Splitting of the Kex2 and Stel3 proteases was carried out analogous to the cloning described in Example Ib. The obtained plasmid was referred to as pSM58.1 (Figure 5). The correct sequence (Seq ID No. 15) was confirmed by sequence analysis. As described for pMHS476.1 in the example le Pichia pastoris KM71H was transformed with the plasmid pSM58.1. The colonies obtained were referred to as Pichia pastoris K 7lH / pSM58. l / a, while "a" represents again the consecutive numbering of the colonies beginning at 1. Example 6a: Insertion of the plasminogen gene Lys from pSM54.2 into the vector pPIC9K 150 mg of the vector pPIC9K (Invitrogen, Groningen, The Netherlands) were cut with every 10 U of the restriction endonucleases Sacl and Notl (both from Roche Diagnostics, Mannheim). 300 ng of the plasminogen expression plasmid pSM54.2 (see example 2b) were also cut with the enzymes Sacl and Notl. The DNA thus treated was separated by gel electrophoresis with a 0.9% agarose gel. In each case the larger fragment was extracted from the gel by means of the QIAgen gel extraction kit (Qiagen, Hilden). The two fragments were combined and ligated at 4 ° C overnight with 1 U of T-DNA-ligase. The transformation of E. coli DH5a, isolation and characterization of the resulting plasmid were carried out analogous to the description in Example Ib, whereas instead of the antibiotic zeocin the antibiotic ampicillin was used for the selection of transformants. The plasmid thus constructed was referred to as pAC37.1 (Figure 6). Example 6b: Transformation of Pichia pastoris with the plasmid pAC37.1 As described for the transformation of Pichia pastoris KM71H with pMHS476.1 in the example le, Pichia pastoris KM71 was transformed with the plasmid pAC37.1 linearized with the restriction endonuclease Sali . The transformed cells were plated on histidine-free medium MD-agar (instruction manual of the Multi-Copy Pichia Expression Kit) and incubated. The colonies obtained were referred to as Pichia pastoris KM71 / pAC37.1, while "a" again represents the consecutive numbering of the colonies beginning at 1. Example 6c: Pichia pastoris culture KM71 / pAC37.1-3 / 1 e induction of the plasminogen gene The production of the precultures and the main cultures as well as the induction with methanol were carried out analogous to the conditions described in the example Id. The induction was carried out on 216 h. It started with a methanol concentration of 0.5%, after 24 h and then in periods of 48 h, 2% methanol was re-fed. Example 6d: Measurement of plasminogen activity in the main culture samples after activation with streptokinase The activity of the plasminogen after activation with streptokinase was determined as described for KM71H / pMHS476. l-l / l to -1/3 in the example le. For the samples taken after 120 h of induction, an activity of 120 U / 1 was obtained. After 216 h of induction, an activity of 190 U / 1 could be measured. Example 6e: Induction of Pichia pastoris KM71 / pAC37.1-3 / 1 in minimal medium (BSM) and measurement of plasminogen activity in the samples of the main cultures after activation with streptokinase After cultivation of Pichia pastoris KM71 / pAC37.1-3 / 1 in BMGY-complex medium (see example Id) 80 g of centrifuged cells were resuspended in 100 ml of minimal medium BSM for the induction phase. The composition of the minimum medium BSM (Basal Sales Medium) is as follows: H3PO485%: 26.0 ml / 1; CaCl2-2H20: 0.6 g / 1; K2S04: 18.0 g / 1; MgSO4-7H20: 14.0 g / 1; KOH: 4.0 g / 1; glycerin: 20 ml / 1; antifoaming: 1.0 ml / 1; tracking solution: 8.0 ml / 1; biotin solution (0.2 g / 1): 8.0 ml / 1. Composition of the screening solution: H2SO4: 5.0 ml / 1; CuS04-5H20: 6.0 g / 1; Kl: 0.08 g / 1; MnS04-H20: 3.0 g / 1; Ma2Mo04: 0.2 g / 1; H3BO3: 0.02 g / 1; CoCl2: 0.5 g / 1; ZnCl2: 20.0 g / 1; FeS04-7H20: 65.0 g / 1. For induction, 2% methanol was added daily. Plasminogen activity after activation with streptokinase was determined as described for KM71H / pMHS476.1-l / l to -1/3 in the example le. After 120 h of induction a plasminogen activity of 193 U / 1 was determined, after 168 h they could be measured 289 U / 1. Example 6f: Detection of plasminogen activity in the main culture samples after activation with streptokinase in the Klárhof fibrinolysis test (open area) For the preparation of the Klárhof fibrinolysis test (open area) (Stack, MS, Pizzo, SV, and Gonzalez-Gronow, M. (1992): Effect of desialylation on the biological properties of human plasminogen, Biochem J. 284, 81-86) (13), 1.5 g of low-melting GTG agarose were fused heating in 75 ml of 50 mM sodium phosphate buffer pH 7.4. 35 ml of fibrinogen solution (225 mg / 37.5 ml 50 mM sodium phosphate buffer pH 7.4) were mixed bubble-free with 350 μ? of thrombin solution (10 U / ml in 50 mM sodium phosphate buffer pH 7.4), were stirred in the agarose solution and emptied into a petri dish. After solidification of the fibrin agar, holes of a size of 1 mm were drilled in the agar. To detect the activity of fibrinolysis of plasminogen produced recombinantly after the activation of streptokinase in each case 20 μ? of the following solutions were pipetted into the holes and incubated for 2Oh at 37 ° C: 1: 0.5 mg / ml of plasminogen (Roche, Mannheim) 2: culture supernatant KM7l / pAC37.1 -3/1 of Example 6e 3: 0.5 mg / ml of plasminogen, activated by streptokinase 4: culture supernatant KM71 / pAC37.1-3 / 1 of example 6e, activated by streptokinase 5: 0.25 mg / ml of plasminogen, activated by streptokinase 6: supernatant of culture KM7l / pAC37.1-3 / 1 of example 6e, diluted 1: 2, activated by streptokinase 7: 0.125 mg / ml of plasminogen, activated by streptokinase 8: culture supernatant KM71H, produced as described in Example 6e for KM7l / pAC37.1-3 / 1, activated by streptokinase For activation with streptokinase 2μ1 of streptokinase (100 U / μ ?, Sigma, Dreisenhofen) were pipetted to 40μ1 of the respective solutions and incubated for 60 min at 37 ° C. The points obtained by fibrinolytic activity are shown in Figure 10. Example 6g: Purification of recombinantly produced plasminogen in Pichia pastoris KM71 / pAC37.1-3 / 1 by 50 ml affinity chromatography of the culture supernatant of Pichia pastoris K 7l / pAC37. l-3 / l of Example 6c / 6d were dialyzed at 4 ° C against 4 1 50 mM sodium phosphate buffer pH 7.5. After 24 h the dialysis buffer was exchanged and dialysed for another 24 h. The dialysate was then pressed through a 0.02 μ? T filter. and then emptied into a TM 4B lysine-sepharose column (diameter: 16 mm, height: 95 mm) (Amersham Biosciences) equilibrated with 50 mM sodium phosphate buffer pH 7.5. Non-specifically bound proteins were washed from the column with 50 mM sodium phosphate buffer pH 7.5, 0.5 M NaCl. The bound plasminogen was eluted with 50 mM sodium phosphate buffer pH 7.5, 0.01 M e-aminocaproic acid. Individual samples were analyzed by 7.5% SDS-PAGE with subsequent silver staining (Figure 11). The recombinant plasminogen contained in the fractions is located in the gel at the height of the human plasminogen added as a reference. Figure 11 shows 7.5% SDS-PAGE of the purification fractions of Example 6g. In Figure 11 the abbreviations used have meanings as follows: M: standard size (from top to bottom: 116 kDa, 66 kDa, 45 kDa, 35 kDa), D: dialysate, N: unbound fraction,: wash fraction, F1-F5 elution fractions containing plasminogen, Plg: plasminogen (American Diagnostica, Pfungstadt) Example 6h: Fermentation of Pichia pastoris K 71 / pAC37.1-3 / 1 for the evaluation of the pH value and the influence of the substrate 50 ml of YEP-G medium (10 g / 1 of yeast extract, 20 g / 1 casein peptone, 20 g / 1 glycerol) in a 1 1 wide neck flask without baffles was inoculated with 2 ml of cryo-culture Pichia pastoris KM71 / pAC37-3 / 1 glycerol and incubated for 9 to 30 ° C and 300 rpm. 5 ml of this culture were used to inoculate 50 ml of MG medium (5 g / 1 of yeast nitrogen base with / without amino acids, 30 g / 1 of glycerol, 2.5 ml / 1 of biotin solution (0.2 g / 1 ) in a 1 1 wide-neck shake flask without baffles This second preculture was incubated for 16 h at 30 ° C and at 300 rpm The main culture was fermented in the multi-fermentation apparatus "stirrer-pro" (DASGIP, JüLICH ), which allows the parallel fermentation of four crops in different conditions, and in each case 150 ml of BSM medium was inoculated (see example 6e) and inoculated with 15 ml of the second pre-culture, the fermentations started at a pH of 6, The target pH value was stopped after starting the dosing of the substrate The different conditions and results of the parallel fermentations are shown in tab 1. Tab 1: Exp. pH substrate feed rate OD600 plasminogen concentration IS methanol profile 187 1.4 mg / 1 II 7 methanol profile 160 6.1 mg / 1 III 6 methanol / glycerol 1 ml / h 270 10.1 mg / 1 IV 6 methanol profile 130 3.4 mg / 1 In experiment IV, 30 g was added to the medium / 1 peptone. Before the start of methanol dosing, the glycerol feed medium (500 g / 1 water-free glycerol, 10 ml / 1 tracer solution, 10 ml / 1 biotin solution [see example 6e]) was added. for 4 h with a constant rate of 24 ml / h. For the profile in experiments I, II and IV the following term was given as a dosing function f (x) = P1 + (P2 / l + exp (-P3 (t-P4)))) + (P5 / (l + exp (-P6 (t-P7)))) with P1 = 0; P2 = 0.7; P3 = 0.2; P4 = 15; P5 = P6 = P7 = 0. It can be seen from tab. 1, that the concentrations of the plasminogen at a neutral pH value and the mixed dosages of glycerol / methanol are the highest. Example 7a: Insertion of the plasminogen Lys gene of pAC37.1 into the vector pGAPZotA 150 ng of the vector pGAPZaA (Invitrogen, Groningen, The Netherlands) were cut with each 10 U of the restriction endonucleases Xhol and Notl (both from Roche Diagnostics, annheim). 300 ng of the plasminogen expression plasmid pAC37.1 (see example 6a) were also cut with the enzymes Xhol and Notl. The DNA thus treated was separated by gel electrophoresis with a 0.9% agarose gel. The 2715 bp fragment of the plasminogen gene from pAC37.1 as well as the 3073 bp fragment of the vector from pGAPZ A were extracted from the gel by means of the QIAgen gel extraction kit (Qiagen, Hilden). The two fragments were combined and ligated at 4 ° C overnight with 1 U of T4 -ADN-ligase. The transformation of E. coli DH5a, the isolation and the characterization of the resulting plasmid were carried out analogous to the description in Example Ib, although instead of the antibiotic zeocin the antibiotic ampicillin was used for the selection of the transformants. The plasmid thus constructed was referred to as pJ 9.1 (Figure 7).

Example 7b: Transformation of Pichia pastoris with the plasmid pJW9.1 As described for the transformation of Pichia pastoris K 71H with pMHS476.1 in the example le, Pichia pastoris K 71H was transformed with the plasmid pJW9.1 linearized with the endonuclease of Blnl restriction. Transformed cells were plated on YPDS agar with 100 μg / ml zeocin (EasySelect ™ Pichia Expression Kit instruction manual) and incubated. The colonies obtained were referred to as Pichia pastoris K 71H / pJW9.1-a, while "a" represents the consecutive numbering of the colonies beginning at 1. Example 7c: Fermentation of Pichia pastoris KM7IH / pJW9.1-3 for the evaluation of the pH value at the glycerol feed rate Pre-cultures and fermentation in the "stirrer-pro" were carried out as described in Example 6 i.

The results are shown in tab. 2. Tab. 2: Exp. PH substrate feed rate OD600 conc. of plasminogen I 6.5 glycerol 1 ml / h 220 18.6 mg / l II 7.0 glycerol 1 ml / h 203 22.2 mg / l III 6.5 glycerol 0.5 ml / h 142 10.1 mg / l IV 7.0 glycerol 0.5 ml / h 99 3.8 mg / l Also in In the case of the glycerol feed, the best yields were obtained by fermentation at a neutral pH value, although the influence of the substrate dosage (feed rate) on the formation of the product can be clearly seen. Example 7d: Fermentation of Pichia pastoris K 71H / pJW9.1-3 50 ml of YEP-G medium (10 g / 1 yeast extract, 20 g / 1 casein peptone, 20 g / 1 glycerol) in a flask 1 1 wide-neck without baffles, were inoculated with Pichia pastoris K 71H / pJW9.1-3 and incubated for 9 h at 30 ° C and at 300 rpm. 10 ml of this culture were used to inoculate 40 ml of MG medium (5 g / 1 of yeast nitrogen base with / without amino acids, 30 g glycerol, 2.5 ml / 1 of biotin solution (0.2 g / 1)) in a 1 1 wide-necked shake flask without baffles. This culture was incubated for 16 h at 30 ° C and at 300 rpm. 3 1 of BSM medium (see example 6e) were inoculated with 30 ml of this culture in a laboratory thermower of 7.5 1 (type Labfors, Infors AG, CU). The fermentation was carried out at 25 ° C and a constant gas feed rate of 3.2 1 / min. After 24 h, glycerol solution (500 g / 1 glycerol, 10 ml / 1 tracer solution, 10 ml / 1 biotin solution [see example 6e]) was added. The dosage rate was increased stepwise from 10 ml / h to 45 ml / h during fermentation. After 250 h, an inorganic plas activity of 1375 U / 1 could be measured after activation with streptokinase. Example 8: Identification of commercially available plasminogen activators 24 proteases were tested for their eligibility for plasminogen activation. The experiments for the same were carried out in 100 mM sodium phosphate buffer pH 8, 0.36 mM CaCl2, 0.9% NaCl. The proteases supplied in the form of powders were dissolved in buffer, the proteases supplied in solution were used directly and respectively diluted with buffer as needed. 25 μ? of the protease solutions were mixed with 25 μ? of plasminogen according to the present invention (20 mg / ml) and incubated for 10 min at 37 ° C. After this the plasmin activity was measured with respect to the N-tosyl-Gly-Pro-Lys-pNA substrate. For this, 200 μ? of substrate solution (9.5 mg of N-tosyl-Gly-Pro-Lys-pNA, dissolved in 75 mg of glycine / 10 ml, 2% Tween® 20) were pipetted at 850 μ? of buffer, were merged with the 50 μ? of the pre-incubated plasminogen protease mixture and further incubated at 37 ° C. The increase in the extinction was measured photometrically at 405 nm. To measure the increase in the extinction due to the proteases, tests were carried out in which, instead of the pre-incubated plasminogen protease mixture, a mixture of pre-incubated buffer protease was used in a similar manner. S. griseus protease, protease VIII, protease XXIII, protease XIX, protease XVIII, ficin, metalloendopeptidase, clostripain, Glu-C, protease XIII, cymopapain, cimothyrosine, protease X, bromelain, kallikrein and proteinace A were purchased from Sigma, Deisenhofen; Trypsin, papain, Asp-N, dispase 1, Lys-C, thrombin and elastase came from Roche, Annheim; Proteinase K was supplied by QIAGEN, Hilden. The protease storage solutions produced had the protein concentrations given in Table 3. The dilution factor F indicates at what proportion the storage solutions are diluted for the measurements. Then the plasmin activities could be determined after activation (1 U / mg = 1 μt N-tosyl-Gly-Pro-Lys-pNA conversion per minute per mg of protein): Table 3: Protease activity of plasmin conc , of protein F after activation [mg / ml] Protease of S. griseus 613.3 U / mg 0.77 1000 Protease VIII 9 U / mg 3.58 1000 Protease XXIII 17.8 50000 Protease XIX 2.78 100 Protease XVIII 0.7 U / mg 1.79 100 Ficin 0.01 U / mg 0.81 1 Metalloendopeptidase 8.9 U / mg 0.01 1 Clostripain 1.7 U / mg 0.25 1 Endoproteinase Glu-C 0.6 U / mg 0.81 1 Protease XIII 0.01 U / mg 0.43 1 Ciraopapain 2.02 1 Cimotripsin 0.14 1 Protease X 2.01 1 Brome1ain 0.81 1 Kallikrein 0.56 1 Protein A 0.02 U / mg 0.36 1 Trypsin 11 kU / mg 3.40 100000 Papain * 0.64 10 Endoproteinase Asp-N 4.3 U / mg 0.004 1 Dispase 1 * 0.2 1 Endoproteinase Lys-C * 0.01 1 Thrombin 83.0 U / mg 0.59 500 Elastase 0.63 U / mg 0.36 5 Proteinase K * 3.60 100 * For proteases, protease XXIII, protease XIX, cymopapain, endoproteinase Lys-C, cymotrypsin, papain, dispase 1, protease X, bromelain, kallikrein and proteinace K, none could be detected. activation of plasminogen. Example 9: Pharmaceutical Formulations The recombinant functional plasminogen used in the following examples was obtained by the inventive production method. In this respect the term "plasminogen" refers to the micro-, mini-, Lys- or recombinant Glu plasminogen and the term "plasmin" to plasmin which was obtained by the proteolytic cleavage of the micro-, mini-, Lys- or recombinant Glu plasminogen. Activation of the micro, mini-, Lys- or Glu plasminogen can be obtained by using the same plasminogen activators, especially the proteases that activate the plasminogen as described above, but is not limited to these examples, although the proportion of Activator units to plasminogen units (micro-, mini-, Lys-, or plasminogen Glu) is approximately 1: 1000.

Plasminogen can be proteolytically activated, i.e., by the tissue plasminogen activator of proteases, urokinase or the proteases protease VII or the protease of S. griseus described in the patent as well as by complex formation with streptokinase or staphylokinase. Example 9a: Pharmaceutical formulations Hydrogels Base formulation for hydrogels (lOOg) Plasminogen 100 U Plasminogen activator (s) 0.1 U Hydroxyethyl cellulose 10 000 3.5 g optional preservation (sorbic acid / potassium sorbate 0. 1-0.4%, PHB-ester 0.1%) purified water ad 100.0 The resp. of methyl cellulose hypromellose instead of resp. of hydroxyethyl cellulose in an amount of 0.5-15.0 g. Plasminogen gel 1000 U Activator (s) of plasminogen 1 U Glycerol (85%) 150.0 g Hydroxyethyl cellulose 10 000 32.5 g optional storage (sorbic acid / potassium sorbate 0.1-0.4%, PHB-ester 0.1%) Ringer's solution without lactate ad 1000.0 g alternatively: 100 g contain: Plasminogen 100 U Plasminogen activator (s) 0.1 U Hydroxyethyl cellulose 10 000 2.5 g Glycerol 85% 10.0 g optional preservation (sorbic acid / potassium sorbate 0.1-0.2%, PHB-ester 0.1% ) water purified ad 100.0 alternatively: 100 g of gel contain: Plasminogen 100 U Plasminogen activator (s) 0.1 U Polyacrylic acids 1 g Propylene glycol 8 g Mid-cathexed triglyceride 8 g Diethylamine (to adjust the pH) qs optional storage (sorbic acid / potassium sorbate 0.1-0.2%, PHB-ester 0.1%) 2-Propanol 0-1 g Water ad 100 g Hydrophilic ointment (Macrogol ointment) 50g contains Plasminogen 50 U Plasminogen activator (s) 0.05 U Macrogol 400 30.0 g Macrogol 4000 10.0 g optional storage (sorbic acid / potassium sorbate 0.1-0.2%, PHB-ester 0.1%) Purified water ad 50.0 g alternatively: Water-free Macrogol ointment 100 g contain: Plasminogen 100 U Activator (en) of the plasminogen 0.1 U Macrogol 300 50 g Macrogol 1500 ad 100 g alternatively: Water reabsorbent ointment Plasminogen 100 U Plasminogen activator (s) 0.1 U Cetilestearyl alcohol 29 g Paraffin, viscose 34 g Vaseline, white 100 g Hydrophobe Plasminogen Ointment 100 U Plasminogen activator (s) 0.1 U Vaseline 80.0 g Slim paraffin fluid ad 100 g Plasminogen 100 U hydrophobic paste U Plasminogen activator (s) 0.1 U Hypromellose 400 20 g Vaseline, white ad 100 g alternatively: Plasminogen 100 U Plasminogen activator (s) 0.1 U Carbomer. { eg, carbopol 974p) 15 g Paraffin, viscose 40 g White petrolatum ad 199 g Plasminogen cream 100 U Plasminogen activator (s) 0.1 U Mid-caged triglycerides 20 g Cetyl stearyl alcohol emulsifier 10 g Lanolin 10 g Sorbitol 10 g optional preservation (acid sorbican / potassium sorbate 0.1-0.2%, PHB-ester 0.1%) purified water ad 100 g Non-ionic hydrophilic cream Plasminogen 100 U Plasminogen activator (s) 0.1 U Cetyl alcohol 20 g 2 -Ethylauromyristate 10 g Glycerol 85% 6 g Potassium sorbate 0.14 g Citric acid 0.07 g Water ad 100 g Non-ionic cream Plasminogen 100 U Plasminogen activator (s) 0.1 U Polysorbate 60 5 g Cetylstearyl alcohol 10 g Glycerol 85% 10 g Vaseline, white 25 g optional preservation (sorbic acid / potassium sorbate 0.1-0.2%, PHB-ester 0.1%) Water ad 100 g Liposomal Formulation Plasminogen 100 U Activated (s) of plasminogen 0.1 U Soy lecithin, chicken lecithin 15 g conservation option al (sorbic acid / potassium sorbate 0. 1-0.2%, PHB-ester 0.1%, resp. diazodinil urea 1-2 g) Water ad 100.0 g Capsule One capsule with 0.25 g powder / granulate contains: Plasminogen 5 U Plasminogen activator (s) 0.005 U Starch 0.1 g Silicon dioxide 0.02 g Magnesium stearate 0.002 g Polymethyl methacrylate copolymers / polymethacrylic acid 0.015 g Triethylcitrate 0.0005 g Talc 0.001 g Cellulose, microcrystalline ad 0.25 g alternately: One capsule with 0.25 g powder / granulate contains: Plasminogen 5 U Plasminogen activator (s) 0.005 U Silicon dioxide 0.01 g Magnesium stearate 0.002 g Copolymerized polymethacrylate / polymethacrylic acid 0.015 g Triethylcitrate 0.0001 g Talcum 0.001 mg Mannitol ad 0.25 g Pills 100 mg pill pellets contain: Plasminogen 5 U Plasminogen activator (s) 0.005 U Starch 30 mg Silicon dioxide 2 mg Magnesium stearate 4 mg Polymethacrylate / polymethacrylic acid copolymerisates 5 mg Triethyl citrate 0-1 mg Talc 0.0001 mg Cellulose , microcrystalline ad 100 mg Pills 100 g tablets contain: Plasminogen 2000 U Plasminogen activator (s) 2 U Starch 20 g Sucrose stearate 20 g Silicon dioxide 2 g Magnesium stearate 3 g Polyvinylpyrrolidone 0-1 g Polymethacrylate copolymerisates / polymethacrylic acid 5 g Talc 0.2 g Triethyl citrate 0.1 g Cellulose, microcrystalline ad 100 g Solution for injection Plasminogen 500 U Plasminogen activator (s) 0.5 U Ethanol 0-1 g Propylene glycol 10 g Polyethylene glycol 0-1 g Sodium chloride qs optional preservation (phosphate sodium hydrogen / sodium dihydrogen phosphate 0.1-0.2%, PHB-ester 0.1%) Purified water ad 100 ml Instead of micro- , mini-, Lys- or Glu plasminogen in the case of the listed formulations the same amount can also be used based on plasmin activity. If plasmin is used directly, no plasminogen activator has to be contained in the pharmaceutical formulation. Example 9b: Pharmaceutical formulations a) Hydrogels Basic formulation for hydrogels (100 g) Plasmin 100 U Hydroxyethylcellulose 400 2.5-5.0 g purified water ad 100.0 g The time for dilation takes from 1 to 3 hours. It is possible to use 1-1000 U of plasmin per gram of hydrogel. b) Hydrophilic ointment Basic formulation of a hydrophilic ointment (1000 g): Plasmin 1000 U Glycerol, water-free 85.0 g Hydroxyethylcellulose 10,000 32.5 g Optionally 0.2% polyhexanide by weight Ringer's solution without lactate ad 1000.0 g Can. optionally added polyhexanide as active anti-microbial agent in a concentration of up to 0.2% by weight. Hydroxyethylcellulose 400 [e.g., Tylose® H 300 or Natrosol 250® HX PHARM) can also be added instead of 10,000 hydroxyethylcellulose (Natrosol 250® HX PHAR). It is possible to use 1-10000 U of plasmin per gram of ointment, (c) Ointment Basic Formulation for ointment (50 g) Plasmin 50 U Macrogol 400 30.0-32.5 g Macrogol 4000 12.5-7.5 g purified water ad 50.0 g Preparation: 12.5 g of macrogol 4000 and 30.0 g of macrogol 400 (in the case of ointments it supplies 7.5 g of macrogol 4000 and 32.5 g of macrogol 400) are heated in the water bath in an ointment tank until the macrogol is smelled. After cooling the appropriate amount of plasmin, which was produced by the inventive method, it is added dissolved in 7.5 g of purified water and thereafter homogenized. d) Capsule Basic formulation for 0.5 g Plasmin 5 U Lactose 0.42 g Starch 0.06 g Magnesium stearate 0.02 g It is possible to use 0.1-100 U of plasmin per capsule. e) Injectable solution / infusion solution Basic formulation for 100 ml Plasmin 500 U Ethanol 0.01 g Polypropylene glycol 30 ml purified water ad 100 ml It is possible to use 1-500 U of plasmin per ml of solution. Instead of plasmin, micro-, mini- Lys- or Glu plasminogen can also be used in the amounts mentioned for plasmin on the basis of the activity in units, if at the same time at least one plasminogen is added in an amount of 1: 10000 to 1: 100, preferably in an amount of 1: 1000 based on the activity of the plasminogen. Example 10a: Amplification and cloning of different forms of the mini- and micro-plasminogen gene and cloning into the vector pPICZaA; transformation of Pichia pastoris The mini- and micro-plasminogen represent shortened plasminogen derivatives, which lack the N-terminal domains, but which can still be activated within the active plasmin. The amplification of the mini- and micro-plasminogen genes for cloning into the vector pPICZaA was carried out with the oligonucleotide primer N034 for the 3 'end and in each case with one of the primers N036a-j (Seq ID No 19 to 28) for the particular 5 'end using the conditions described in example la. The oligonucleotide primers N036a, c, e, g, i have, in addition to the bases complementary to the plasminogen gene, the codons for the cleavage site Kex2, the primers N036b, d, f, h, j have in addition subsequent codons for the cleavage site Kex2 the codons for two cleavage sites Stel3. The initiator N034 also has in a cleavage site Kspl, the primers N036 a-j have a cleavage site X ol. The cloning of the mini-and micro-plasminogen genes in the pPICZaA vector was carried out analogous to the cloning described in Example Ib, although the vector as well as the particular PCR product were cut with the restriction endonucleases Xhol and Kspl . The primers used, the names of the plasminogen derivative, the encoded protease cleavage sites, the labeling of the obtained plasmids and the N-terminal amino acid of the secreted plasminogen derivative are summarized in the following table.

Initiator initiator name point name Amino acid * 5 '3' cleavage of the N-terminal protease plasmid N03Sa N034 mini- laminogen Kex2 pPLGl. 1 A463 N036b N034 mini-plasminogen Kex2,2xStel3 pPLG2. 1 A463 N03Sc N034 Kex2 micro-plasminogen pPLG3. 2 K550 N03Sd N034 micro-plasminogen Kex2,2xStel3 pPLG4. 2 K550 ? 036T N034 micro-plasminogen ex2 pPLG5. 3 L551 N036f N034 Kex2 micro-plasminogen, 2xStel3 pPLG6. 1 L551 N036g N034 Kex2 micro-plasminogen pPLG7. 1 A562 N036h N034 micro-plasminogen Kex2, 2xStel3 pPLG8. 3 A562 N036j N034 micro-plasminogen ex2 pPLG9. 1 S564 N036j N034 micro-plasminogen ex2, 2xStel3 pPLGIO. 1S564 * The numbering refers to the preplasminogen of 810 amino acids in length (Seq ID No. 12) Figure 8 shows exemplary plasmid pPLGl .1. As described for pMHS476.1 in the example, Pichia pastoris was transformed with the plasmid pPLGl .1. The colonies obtained were referred to as Pichia pastoris KM71H / pPLGl .1-l / a, while "a" represents the consecutive numbering of the colonies beginning at 1.

The generation of strains based on plasmids pPLG2.1, pPLG3.2, pPLG .2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1, and pPLGIO .1 was carried out according to the production of strain K 71H / pPLGl .1-l / a. Oligonucleotide primer N36a-j N036a AAAAACTCGAGAAAAGACCACCTCCGCCTGTTG N036b AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG N036C AAAAACTCGAGAAAAGAAAACTTTACGACTACTG N036d AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG N036e AAAAACTCGAGAAAAGACTTTACGACTACTGTG N036f AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG N036g AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG N036h AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG N036Í AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC N036j AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC Example 10b: Amplification and cloning of different forms of the mini-gene and micro-plasminogen and cloning within the pGAPZaA vector transformation of Pichia pastoris Amplification of the mini- and micro-plasminogen genes for cloning into the pGAPZaA vector performed with the oligonucleotide primer N034 for the 3 'end and in each case with one of the primers N036a-j (Seq ID No. 19 to 28) for the particular 5' end using the conditions described in Example la. The oligonucleotide primers N036a, c, e, g, i have, in addition to the bases complementary to the plasminogen gene, the codons for the cleavage site Kex2, the primers N036b, d, f, h, j have in addition subsequent codons for the cleavage site Kex2 the codons for two cleavage sites Stel3. The initiator N034 also has in a cleavage site Kspl, the primers N036 a-j have a cleavage site Xhol. The cloning of the mini- and micro-plasminogen genes in the vector pGAPZaA was carried out analogous to the cloning described in Example Ib, although the vector as well as the particular PCR product were cut with the restriction endonucleases Xhol and Kspl . Summing up the initiators, the names of the plasminogen derivative, the encoded protease cleavage sites, the labeling of the obtained plasmids and the N-terminal amino acid of the secreted plasminogen derivative can be taken from the following table.

Initiator initiator name point name Amino acid 5 '3' cleavage of the N-terminal protease plasmid N036a N034 mini- laminogen Kex2 pPLGll. .1 A463 NO36b N034 mini-Kex2 plasminogen, 2xStel3 pPLG12. .1 A463 N036C N034 Kex2 micro-plasminogen pPLG13. .2 K550 N03Sd N034 micro-plasminogen ex2, 2xStel3 pPLG14. .2 K550 N036e N034 meiero-plasminogen Kex2 pPLG15, .3 L551 N03Sf N034 micro-plasminogen Kex2, 2xStel3 pPLG16. .1 L551 N036g N034 micro-plasminogen ex2 pPLG17..1 A562 N036h N034 micro - plasminogen Kex2,2xStel3 pPLG18. .3 A562 N036j N034 micro-plasminogen Kex2 pPLG19. .1 S564 N036j N034 micro-plasminogen ex2.2xStel3 pPLG20. .1 S564 * The numbering refers to the preplasminogen of 810 amino acids in length (Seq ID No. 12) Figure 9 shows exemplary plasmid pPLGll .2. As described for pJW9.1 in example 7a Pichia pastoris K 71H was transformed with the plasmid pPLGll.l was linearized by the restriction endonuclease Blnl. The obtained colonies were referred to as Pichia Pastoris KM71H / pPLGll .2-l / a, while "a" again represents the consecutive numbering of colonies beginning at 1. Generation of strains based on plasmids pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2, and pPLG20.1 was carried out according to the production of strain KM71H / pPLGl .1- the.

Sequence Protocol Sequence 1: Oligonucleotide primer N034 AAAAACCGCGGTCAATTATTTCTCATCACTCCC Sequence 2: Oligonucleotide primer N036 AAAAACTCGAGAAAAGAAAAGTGTATCTCTCAGAGTG Sequence 3: Oligonucleotide primer N057 AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTG Sequence 4: Oligonucleotide primer N037 AAAAATTCGAAAAATGGAACATAAGGAAGTGG Sequence 5: Oligonucleotide primer N035 AAAAACTCGAGAAAAGAGAGCCTCTGGATGACTAT Sequence 6: Oligonucleotide primer N056 AAAAACTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTAT Sequence 7: human Lys plasminogen fusion gene with the codons for the Kex2 cleavage site and the yeast alpha factor signal gene 3aecharomyees cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTAC AGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCT CCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTAC TGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAG AGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAA AACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCT CAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAG AATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAAC AAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCC ACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTT TCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCA GAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGG GCCCCATGGTGCCATACAACCAAC AGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCC TGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACC CCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACC ACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAG ACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCC GATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTG AAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCA GATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGC AAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCAT AGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGC CGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTT TACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAA GTGGAGCCGAAGAAATOTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCA TCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAA ATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTG CCTTGCTAAAGCTA AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTAT GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTT GGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGC TATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGT GTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 8: Lys human plasminogen with the Kex2 cleavage site and the signal peptide of the alpha factor yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRKVYLSECKTGNGK Y RGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQG PWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDS QSPHAHGYIPSKFPN NLKK YCR PDRELRPWCFTTDPN RWELCDIPR CTTPPPSSGPTYQCLKGTGE YRG VAVTVSGHTCQHWSAQTPHTHNRTP ENFPCK LDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQ LAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQK TPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASV VAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPH RHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCA APSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTL ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEV LEPHVQEIEVSRLFLEP TRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITG GETQGTF GAGLL EAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG GPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMR N * Sequence 9: human Lys plasminogen fusion gene with the codons for the cleavage site Kex2 and two cleavage sites Stel3 and the gene for the signal sequence of the alpha factor of the yeast Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTGCAAGACTGGGAAT GGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGG AGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTG GAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACT GATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCAT TGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAG GCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAG AACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACC ACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCA TCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTG GCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACAT AACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCT GACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGT AAGATACCGTCCTGTGACTCCTC CCCAGTATCCACGGAACAATTGGCTCCCACAGCACCA CCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGC ACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACAC CGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGG AATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAG TACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTT GTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAA GGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCC CAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAA AAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAAT CCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGT GGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCC CCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC CCAGCTTGTCTGCCA TCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC CAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAA GTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGG CATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTC GAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATG AGAAATAATTGA Sequence 10: human Lys plasminogen with the cleavage sites Stel3 and Kex2 and the signal peptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPV TTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTN GLLFINTTIASIAAKEEGVSLEKREAEAKVYLSECKTGN G NYRGTMSKTK GITCQKWSSTSPH PRFSPATHPSEGLEE YC NPDN DPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKT SGLECQ A DSQSPHAHGYIPSKFPN LKKNYCR PDRELRPWCFTTDPNKRWELC DIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTH RTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPV STEQLAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTG KCQSWSSMTPH RHQKTPENYPNAGLTM YCR PDADKGP CFTTDPSVRWEYC LKKCSGT EASWAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAA QEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPR LYDYCDV PQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFC GGTLISPEWVLTAAHCLEKSPRPSSY VILGAHQEVNLEPHVQEIEVSRL FLEPTR DIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGET QGTFGAGLLKEAQLPVIENKVCMRYEFLNGRVQSTELCAGHLAGGTDSCQ GDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVM RN * Sequence 11: human pre-laminogin gene ATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTCAAGGAGAG CCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAGCAG CTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACC TGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAACAGG AAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTATCTC TCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAAT GGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCT ACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAG GGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAG TGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACC ATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG CTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAA TCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT GGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT ACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 12: human preplasminogen MEHKEWLLLLLFLKSGQGEPLDDYV TQGASLFSVTKKQLGAGSIEECA AKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIII MRD LFEK Vyl SECKTGNGKNYRGT SKTK GITCQKWSSTSPHRPRFSPATHPSEGLEEN YCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKT SGLECQAWDSQSPHAHGYIPS FPNK LKK YCRNPDRELRPWCFTTDP NKRWELCDIPRCTTPPPSSGPTYQCLKGTGE YRGNVAVTVSGHTCQHWS AQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIP SCDSSPVSTEQLAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGKKCQS WSSMTPHRHQKTPENYPNAGLTM YCRNPDADKGP CFTTDPSVRWEYCN LKKCSGTEASWAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGT PCQDWAAQEPHRHSIFTPETNPRAGLEK YCR PDGDVGGPWCYTTNPRK LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRT RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSY VILGAHQEV LEPHVQ EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECF ITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA GGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV TWIEGVMR N * Sequence 13: human Glu plasminogen fusion gene with the codons for the Kex2 cleavage site and the gene for the alpha factor signal of the yeast Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAGAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTC AGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAG GAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTG ATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTT GAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACG ATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGA CCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAAT CCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGAC TACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGAC GGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCA CACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGT CGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGG GAACTTTGCGACATCCCCCGCTGC ACAACACCTCCACCATCTTCTGGTCCCACCTACCAG TGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCAC ACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTC CCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGG TGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCC TCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTC CAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACA GGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAA AACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGC CCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGC TCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAG ACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCG ACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGC ATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCT GATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTAC TGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCG AAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGG CAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCA GAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCT GCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCT GACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC AGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTA CAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 14: Human Glu plasminogen with the cleavage site Kex2 and the signal peptide of the alpha factor of the yeast Saccharomyces cerevisiae FPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNST NGLLFINTTIASIAAKEEGVSLEKREPLDDYVNTQGASLF SVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSI IIRMRDWLFEKKVYLSECKTGNGK YRGTMSKTKNGITCQKWSSTSPHR PRFSPATHPSEGLEE YCRNPDNDPQGP CYTTDPEKRYDYCDILECEEE CMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNK LKK YC RNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYR G VAVTVSGHTCQH SAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPW CHTTNSQVR EYCKIPSCDSSPVSTEQLAPTAPPELTPWQDCYHGDGQS YRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKG PWCFTTDPSVRWEYCNLKKCSGTEASWAPPPWLLPDVETPSEEDCMFG NGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEK YCR P DGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGG CVAHPHSWP QVSLRTRFGMHFCGGTLISPE VLTAAHCLEKSPRPSSYK VILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPA CLPSPNYWADRTECFITG GETQGTFGAGLLKEAQLPVIENKVCNRYEF LNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGC ARPNKPGVYVRVSRFVTWIEGVMRNN * Sequence 15: fusion gene plasminogen Glu human with codons for site splitting Kex2 and two cleavage sites Stel3 and the gene for the alpha factor signal sequence of the yeast Saccharomycea cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTATGTGAATACCCAGGGG GCTTCACTGTTCAGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCA GCAAAATGTGAGGAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAG CAACAATGTGTGATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGAT GTAGTTTTATTTGAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAAC TACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACT TCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAAC TACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAA AAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGA GAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGAC TCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAG AAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCC AACAAGCGCTGGGAACTTTGCGAC ATCCCCCGCTGCACAACACCTCCACCATCTTCTGGT CCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACC GTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACA CCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAA AGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCG TCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTA ACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCC ACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAG AAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGAT GCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAAC CTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTT CCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGA GGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCC CATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTAC TGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAA CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTG GCCCACCCACAT TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACC TTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG CTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAAT TATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCT TGA GGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT Echelon 16: Human Glu plasminogen with Stel3 and Kex2 cleavage sites and the yeast alpha factor signal peptide Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPV TTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNST NGLLFINTTIASIAAKEEGVSLEKREAEAEPLDDYVNTQG ASLFSVTKKQLGAGSIEECAA CEEDEEFTCRAFQYHSKEQQCVIMAENR KSS11IRMRDWLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSST SPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILE CEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNK LK K YCRNPDRELRPWCFTTDPNKR ELCDIPRCTTPPPSSGPTYQCLKGTG ENYRGNVAVTVSGHTCQHWSAQTPHTH RTPENFPCKNLDE YCRNPDGK RAPWCHTTNSQVR EYCKIPSCDSSPVSTEQLAPTAPPELTPWQDCYHG DGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTM YCR CR PD ADKGPWCFTTDPSVRWEYCNLKKCSGTEASWAPPPWLLPDVETPSEED CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNY PDGDVGGP CYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR WGGCVAHPHS PWQVSLRTRFG HFCGGTLISPEWVLTAAHCLEKSPRP SSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDK VIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCN RYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSW GLGCARPNKPGVYVRVSRF TWIEGVMR N * ! sequence 17: Glu plasminogen sequence (pSM49.8, pSM58.1 and pSM82.1) secreted in the medium EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSK EQQCVIMAENRKSSII IRMRDWLFEKKVYLSECKTGNGKNYRGTMSKTK NGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPD DPQGP CYTTDP EKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQA DSQSPHAHGY IPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSS GPTYQCLKGTGENYRGNVAVTVSGHTCQH SAQTPHTHNRTPENFPCKNL DENYCR PDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPE LTPWQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNA GLTMNYCRNPDADKGPWCFTTDPSVR EYCNLKKCSGTEASWAPPPWL LPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPE TNPRAGLEKNYCR PDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGK PQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPE VLT AAHCLE SPRPSSYKVILGAHQEV LEPHVQEIEVSRLFLEPTRKDIALL LSSPAVITDKVIPACLPSPNYWADRTECFITG GETQGTFGAGLLKEA QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEK DKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMR N * quency 18: Sequence Lys plasminogen (p HS476.1, pSM54.2, pAC37.1 and pJW9.1) secreted into the medium KVYLSECKTGNGK YRGTMSKTK GITCQK SSTSPHRPRFSPATHPSEG LEENYCR PD DPQGPWCYTTDPEKRYDYCDILECEEECMHCSGE YDGK ISKTMSGLECQAWDSQSPHAHGYIPSKFPNK LKKMYCRNPDRELRPWCF TTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTC QHWSAQTPHTH RTPENFPCKNLDE YCR PDGKRAPWCHTTNSQVR EY CKIPSCDSSPVSTEQLAPTAPPELTPWQDCYHGDGQSYRGTSSTTTTGK KCQSWSSMTPHRHQKTPENYPNAGLTMNYCR PDADKGPWCFTTDPSVRW EYCNLKKCSGTEASWAPPPWLLPDVETPSEEDCMFGNGKGYRGKRATT VTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCR PDGDVGGPWCYTT NPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQV SLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEV LE PHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADR TECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCA GHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRV SRFVTWIEGVMRNN * cuencia 19: Oligonucleotide primer N036a AAAAACTCGAGAAAAGAGCACCTCCGCCTGTTG cuencia 20: Oligonucleotide primer N036b AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG Sequence 21: Initiator oligonucleotide N036c AAAAACTCGAGAAAAGAAAACTTTACGACTACTG sequence 22: Initiator oligonucleotide N036d AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG sequence 23: Initiator oligonucleotide N036e AAAAACTCGAGAAAAGACTTTACGACTACTGTG sequence 24: Initiator oligonucleotide N036f AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG sequence 25: Initiator oligonucleotide N036g AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG sequence 26: Initiator oligonucleotide N036h AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG sequence 27 : Oligonucleotide primer N036i AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC Sequence 28: Oligonucleotide primer N036j AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC Sequence 29: Mini-plasminogen (pPLGl .1 and pPLG2.1) APPPWLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHR HSIFTPETNPRAGLEKNYCRNPDGDVGGP CYTTNPRKLYDYCDVPQCAA PSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFG HFCGGTLI SPE VLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPT RKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITG GETQGTFG AGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGG PLVCFEKDKYILQGVTS GLGCARPNKPGVYVRVSRFVTWIEGVMR N * Sequence 30: Micro-plasminogen (pPLG3.2 and pPLG4.2) KLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLR TRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHV QEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTEC FITG GETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHL AGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRF VTWIEGVMR N * Sequence 31: Micro-plasminogen (pPLG5.3 and pPLG6.1) LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRT RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEV LEPHVQ EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSP YWADRTECF ITG GETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA GGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV TWIEGVMRNN * Sequence 32: Micro-plasminogen (pPLG7.1 and pPLG8.3) APSFDCGKPQVEPKKCPGRWGGCVAHPHS PWQVSLRTRFGMHFCGGTL ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEV LEPHVQEIEVSRLFLEP TRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFI GWGETQGTF GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG GPLVCFEKDKYILQGVTS GLGCARPNKPGVYVRVSRFVTWIEGVMR N * Sequence 33: Micro-plasminogen (pPLG9.1 and pPLGIO.l) SFDCGKPQVEPKKCPGRWGGCVAHPHS PWQVSLRTRFGMHFCGGTLIS PEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTR KDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGA GLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGP LVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN * Sequence 34: DNA sequence of the alpha factor in yeast Saccharomyces cerevisiae pPICZaA to the Kex2 cleavage site.

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTG CTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCAT CGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGC ACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAG AAGGGGTATCTCTCGAG Sequence 35: amino acid sequence of the alpha factor of the yeast Saccharomyces cerevisiae in pPICZaA up to the cleavage site Kex2.

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDF DVAVLPFSNST NGLLFINTTIASIAAKEEGVSLE Sequence 36: DNA sequence of the Kex2 AAAAGA unfolding site Sequence 37: DNA sequence of Stel3 cleavage site GAGGCTGAAGCT Sequence 38: amino acid sequence of the Kex2 KR cleavage site Sequence 39: amino acid sequence of Stel3 cleavage site EAEA Sequence 40: amino acid sequences of human mini-plasminogen as in pPLGl.l with the cleavage site Kex2 and the prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPPPWLLPDVETPS EEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLE KNYCRNPDGDVGGP CYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKC PGRWGGCVAHPHSWP QVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKS PRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVI TDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVIEN VCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGV TSWGLGCARPNKPGVYVRVSRFVT IEGVMRNN * Sequence 41: amino acid sequence of the human mini-plasminogen as in pPLG2.1 with the cleavage site Kex2 and two cleavage sites and the prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV AVLPFSNSTNNGLLFINTTIASIAA EEGVSLEKREAEAAPPPWLLPDV ETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPR AGLEKNYCRNPDGDVGGPWCYTTNPR LYDYCDVPQCAAPSFDCGKPQVE PKKCPGRWGGCVAHPHSWPWQVSLRTRFG HFCGGTLISPEWVLTAAHC LEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSS PAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPV IEN VCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYI LQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN * Sequence 42: amino acid sequence of human plasminogen and micro pPLG3.2 with the Kex2 cleavage site and prepro factor alpha yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYS DLEGDFDVAVLPFSNST GLLFINTTIASIAAKEEGVSLEK RKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPH SWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSS YKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSP AVITDKVIPACLPSPNYWADRTECFITG GETQGTFGAGLL KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQG DSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV TWIEGVMRNN * Sequence 43: amino acid sequence of the human micro-plasminogen as in pPLG4.2 with the cleavage site Kex2 and two cleavage sites Stel3 and the prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPV TTTEDETAQIPAEAVIGYSDL EGDFDVAVLPFSNST NGLLFINTTIASIAAKEEGVSLEKREAE AKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHS P QVSLRTRFG HFCGGTLISPE VLTAAHCLEKSPRPSSYKVI LGAHQEV LEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDK VIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKEAQLPVI ENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFE KDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN * Sequence 44: amino acid sequence of human plasminogen and micro pPLG5.3 with the Kex2 cleavage site and prepro factor alpha yeast Saccharomyces cerevisiae MRFPS IFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDF DVAVLPFSNST NGLLFINTTIASIAAKEEGVSLEKRLYDYCDVPQCA APSFDCGKPQVEPKKCPGRWGGCVAHPHSWPWQVSLRTRFG HFCGG TLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRL FLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITG G ETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGT DSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVT IEGVMRNN * Sequence 45: amino acid sequence of the human micro-plasminogen as in pPLG6.1 with the cleavage site Kex2 and two cleavage sites Stel3 and the prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPV TTTEDETAQIPAEAVIGYSDL EGDFDVAVLPFSNST NGLLFINTTIASI AKEEGVSLEKREAE ALYDYCDVPQCAAPSFDCGKPQVEPKKCPGRWGGCVAHPHSWP WQVSLRTRFGMHFCGGTLISPE VLTAAHCLEKSPRPSSYKVIL GAHQEV LEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKV IPACLPSP YWADRTECFITGWGETQGTFGAGLLKEAQLPVIE NKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEK DKYILQGVTSWGLGCARPNKPGVYVRVSRFVT IEGVMRNN * Sequence 46: Amino acid sequence of human micro-plasminogen as in pPLG7.1 with the cleavage site Kex2 and the prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDF DVAVLPFSNSTN GLLFINTTIASIAAKEEGVSLEKRAPSFDCGKPQV EPKKCPGRWGGCVAHPHS PWQVSLRTRFGMHFCGGTLISPEWVLTA AHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIAL LKLSSPAVITDKVIPACLPSPNYWADRTECFITG GETQGTFGAGLL KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPL VCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN * Sequence 47: Amino acid sequence of the human micro-plasminogen as in pPLG8.3 with the cleavage site Kex2 and two cleavage sites Stel3 and the prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDF DVAVLPFSNST NGLLFINTTIASIAAKEEGVSLEKREAEAAPSFDCG KPQVEPKKCPGRWGGCVAHPHS PWQVSLRTRFGMHFCGGTLISPEW VLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRK DIALLKLSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFG AGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDS GGPLVCFEKDKYILQGVTS GLGCARPNKPGVYVRVSRFVTWIEGVMR N * Sequence 48: Amino acid sequence of the human micro-plasminogen as in pPLG9.1 with the cleavage site Kex2 and the prepropeptide of the alpha factor of the yeast Saccharomyces cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQI AEAVIGYSDLEGDF DVAVLPFSNST GLLFINTTIASIAAKEEGVSLEKRSFDCGKPQVEP KKCPGRWGGCVAHPHS PWQVSLRTRFGMHFCGGTLISPE VLTAAH CLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLK LSSPAVITDKVIPACLPSPNYWADRTECFITGWGETQGTFGAGLLKE AQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVC FEKDKYILQGVTS GLGCARPNKPGVYVRVSRFVTWIEGVMR N * Sequence 49: Amino acid sequence of human plasminogen and micro pPLGIO 1 with Kex2 cleavage site and two cleavage sites Stel3 and prepro alpha factor Yeast Saecharomyees cerevisiae MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDF DVAVLPFSNST GLLFINTTIASIAAKEEGVSLEKREAEASFDCGKP QVEPKKCPGRWGGCVAHPHS PWQVSLRTRFG HFCGGTLISPE VL TAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDI ALLKLSSPAVITDKVI PACLPSPNYWADRTECFITGWGETQGTFGAG LLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGG PLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRN Sequence 50: Nucleic acid sequence of the human mini-plasminogen gene as in pPLGl .1 with the codons for the cleavage site Kex2 and the prepropeptide gene of the yeast alpha factor Saecha omyees cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTG CTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCAT CGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGC ACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAG AAGGGGTATCTCTCGAGAAAAGAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGA GACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGG GCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGAC ACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCG TAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTT TACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTC AAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACA TTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGC ACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAA GGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCA TGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGC CATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGG AGA AACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAG AATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCT GTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCT GGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGC TGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGA TTGAGGGAGTGATGAGAAATAATTGA Sequence 51: Nucleic acid sequence of the human mini-plasminogen gene as in pPLG2.1 with the codons for the cleavage site Kex2 and the Stel3 cleavage sites and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTG CTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCAT CGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGC ACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAG AAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTGTCCTGCT TCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATAC CGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGG AGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAA AAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAAT CCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATT GTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGT GGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCAC TTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGG AGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAA TCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGA AAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCC CAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATC AC TGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTC CCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAAT CCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAG TGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGT TTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 52: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG3.2 with the codons for the ex2 cleavage site and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAA ATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGT GCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAA TGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGG CCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGA GGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTG GAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACAC CAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGG CTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGC AGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCC CCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGG GGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG CTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAAT GGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGC ACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAG AAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGT GCACGCCCCAATAAGCCTG GTGTCTATGTTCGTGTTTCAAGGTTTGTT ACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 53: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG4.2 with the codons for the cleavage site Kex2 and the Stel3 cleavage sites and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAA ATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTGTGAT GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTG GAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCAC CCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATG CACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCT GCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATC CTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATA GAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTG CTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCT TGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTC ATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTAT GAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCAT TTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTG GTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG GGTCTTGGCTGTGCACGCC CCAATAAGCCTGGTGTCTATGTTCGTGTT TCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 54: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG5.3 with the codons for the cleavage site Kex2 and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAA ATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGACTTTACGACTACTGTGATGTCCCTCAGTGTGCG GCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGT CCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCC TGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGC ACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAG AAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAA GAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTG TTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGT CCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCA AATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGA GAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTC CCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGA AGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACT GACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAG GACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCA CGCCCCAATAAGCCTGGTG TCTATGTTCGTGTTTCAAGGTTTGTTACT TGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 55: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG6.1 with the codons for the cleavage site ex2 and the Stel3 cleavage sites and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTG CTCCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCAT CGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGC ACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAG AAGGGGTATCTCTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTGATGTCCC TCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGT CCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCA GTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTG GGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTC ATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGT CTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCC TGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTC GCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAG CTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTA TGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACA AATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAG CC TGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAAT AATTGA Sequence 56: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG7.1 with the codons for the cleavage site Kex2 and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAA ATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTG GAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCAC CCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATG CACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCT GCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATC CTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATA GAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTG CTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCT TGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTC ATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTAT GAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCAT TTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTG GTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTT TCAAGGTTTGTTACTTGGA TTGAGGGAGTGATGAGAAATAATTGA Sequence 57: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG8.3 with the codons for the cleavage site Kex2 and the Stel3 cleavage sites and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAA ATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTGGG AAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGG TGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGG GTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCC TACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCAT GTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAA GATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAA GTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGA GCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTG TGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTC TGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT GGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTC TATGTTCGTGTTTCAAGGT TTGTTACTTGGATTGAGGGAGTGATGAGA AATAATTGA Sequence 58: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG9.1 with the codons for the cleavage site Kex2 and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAA ATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGATCATTTGATTGTGGGAAGCCTCAAGTGGAGCCG AAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACAT TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTC TGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCAC TGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGT GCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG CTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTG CCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACT GGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAA GCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGC TTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTT GGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGG TTTGTTACTTGGATTGAGG GAGTGATGAGAAATAATTGA Sequence 59: Nucleic acid sequence of the human micro-plasminogen gene as in pPLGIO.l with the codons for the cleavage site Kex2 and the Stel3 cleavage sites and the prepropeptide gene of the yeast alpha factor Saccharomyces cerevisiae ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCC GCATTAGCTGCTCCAGTCAACACTACAACAGAAGATGAAACGGCACAA ATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATTG TTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA TCTCTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCCT CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTG GCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTT GGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTG ACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATT GCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC CCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAA TGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCT GGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGT CCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACT TCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT TGA CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT Sequence 60: Nucleic acid sequence of the gene for human mini-plasminogen as pPLGl 1 and pPLG2.1 GCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATG TTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGC CAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCA CGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGG TGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCC CCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTT GGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCAC TGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAA GTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACA CGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC CCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACT GGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCT GTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACC GAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC AGTTGCCAGGGTGACAGTGGAGGT CCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTT GGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGG ATTGAGGGAGTGATGAGAAATAATTGA Sequence 61: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG3.2 and pPLG4.2 AAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTT GATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTT GTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGT CTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCC CCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGG CCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTC GAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCC ACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATC ACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTC GCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGT ACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAG AATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCC ACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAG GGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATT TTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAG CCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGA GTGATGAGAAATAATTGA Sequence 62: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG5.3 and pPLG6.1 CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGAT TGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTT AGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCA GAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAA CCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACA CGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACT GACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCT GACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAAT AAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACC GAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGT GACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTA CAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCT GGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTG ATGAGAAATAATTGA Sequence 63: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG7.1 and pPLG8.3 GCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGT CCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCC TGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGC ACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAG AAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAA GAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTG TTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGT CCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCA AATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGA GAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTC CCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGA AGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACT GACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAG GACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCA CGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACT TGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 64: Nucleic acid sequence of the human micro-plasminogen gene as in pPLG9.1 and pPLGIO.l TCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGA AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAA GTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCC CCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTG AATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTG GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCC GTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTAT GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC CAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTG ATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGT TGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATT GAGGGAGTGATGAGAAATAATTGA Sequence 65: Nucleic acid sequence of the human Glu plasminogen gene GAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAG CAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTC ACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAAC AGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTAT CTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAA AATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCT GCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCG CAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTT GAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAG ACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATAC ATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGG GAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATC CCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACA GGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGG AGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTG GATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAAC AGCCAAGTGCGGTGGGAGTACTGT AAGATACCGTCCTGTGACTCCTCCCCAGTATCCACG GAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCAT GGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAG TCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCT GGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACC ACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCG AGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAA GACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAG ACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGT GGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAG TGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGA AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGA ACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACT GCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCA CACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTG GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGC CGTCATCACTGAC AAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGT TTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCC CAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTT GTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA Sequence 66: Nucleic acid sequence of plasminogen gene Lys h mnao AAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCC AAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGA TTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGAC AACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGC GACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAA ATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCT CATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAAC CCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTT TGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTG AAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGT CAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGC AAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCAT ACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCA GTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGAC TGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAG AAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTAC CCAAATGCTGGCCTGACAATGAAC TACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGG TGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGA ACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCT TCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACT GTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTC ACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGT GATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGAT GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAA TGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTC AGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGG GTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATC CTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGG CTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTC ATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAAT GGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGC CAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTT TCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA Bibliography (1) = Desire Collen, Thrombosis and Haemostasis, 82, 1999 (2) = Forsgren et al., FEBS Lett. 213, 1987 (3) = Petersen et al., J. Biol. Chem., 265, 1990 (4) = Duman et al., Biotechnol. Ap l. Biochem. 28; 39-45, 1998 (5) = Guan et al., Sheng Wu Gong Cheng Xue Bao, 17, 2001 (6) = González-Gronow et al., Biochimica et Biophysica Acta, 1039, 1990 (7) = Whitefleet-Smith et al., Arch. Biochem. Biophys., 271, 1989 (8) = Nilsen und Castellino, Protein Expression and Purification, 16, 1999 (9) = Busby et al., J. Biol. Chem., 266, 1991 (10) = Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989 (11) = Gassen & Schrimpf, Gentechnische Methoden, Spektrum Akademischer Verlag, Heidelberg, 1999 (12) = Malinowski et al., Biochemistry, 23, 1984 (13) = Stack et al., Biochem. J. 284, 1992

Claims (62)

1. CLAIMS 1. A method for the recombinant production of a functional plasminogen in microorganisms comprising at least the step: a) fusion of a nucleic acid sequence encoding at least the functional part of the plasminogen with a nucleic acid sequence coding for at least one signal peptide, the nucleic acid sequence encoding the functional plasminogen and the nucleic acid sequence encoding at least the signal peptide being coupled with the codons for the cleavage sites of the proteases that are provided for the splitting of the signal peptide.
2. 2. The method for recombinant production according to claim 1, wherein the nucleic acid sequence encoding at least the functional part of the plasminogen comprises the proteolytic domain of the plasminogen or a mutant or a fragment thereof.
3. 3. The method for recombinant production according to claim 1 or 2, wherein the nucleic acid sequence encoding at least one functional part of the plasminogen encodes the plasminogen Glu or Lys.
4. 4. The method for recombinant production according to one of claims 1 to 3, wherein the nucleic acid sequence encoding at least one signal peptide encodes a prepropeptide, a prepeptide or a propeptide and / or wherein the codons encode for protease cleavage sites for the Kex2 and / or Stel3 protease.
5. 5. The method for recombinant production according to claim 4, characterized in that the nucleic acid molecule encoding at least one signal peptide codes for the alpha factor signal peptide or SUC2, PHA-E or PH01 of yeast Saccharo yces cerevisiae.
6. 6. The method for recombinant production according to one or more of the previous claims, characterized in that at least 20 mg / l of functional Glu or Lys plasminogen are produced.
7. 7. The method for recombinant production according to one or more of the previous claims, characterized in that after a 120 hour processing at least 120 U / 1 of the functional Lys plasminogen is produced.
8. 8. The method for recombinant production according to one or more of the previous claims, wherein as primers for the amplification two oligonucleotide primers selected from the group consisting of N036a (Seq ID No. 19), N036b (Seq. ID No. 20), N036c (Seq ID No. 21), N036d (Seq ID No. 22), N036e (Seq ID No. 23), N036f (Seq ID No. 24), N036g (Seq ID No. 25) , N036h (Seq ID No. 26), N036Í (Seq ID No. 27), and N036j (Seq ID No. 28).
9. 9. The method for recombinant production according to one or more of the previous claims, wherein as primers for the amplification two oligonucleotide primers selected from the group consisting of N034A (Seq ID No. 1), N036 (Seq. ID No. 2), N057 (Seq ID No. 3), N037 (Seq ID No. 4), N035 (Seq ID No. 5), N056 (Seq ID No. 6), as well as the group consisting of N036a (SEQ ID No. 19), N036b (Seq ID No. 20), N036c (Seq ID No. 21), N036d (Seq ID No. 22), N036e (Seq ID No. 23), N036f (Seq ID No. 24), N036g (Seq ID No. 25), N036h (Seq ID No. 26), N036I (Seq ID No. 27), and N036j (Seq ID No. 28). The method for recombinant production according to one or more of the previous claims wherein the fusion product resulting in step a) is incorporated into the expression vector suitable for microorganisms. The method for recombinant production according to claim 10, wherein the expression vector is suitable for fungi. 12. The method for recombinant production according to claim 10 or 11, wherein the expression vector comprises an inducible and constitutive promoter. 13. The method for recombinant production according to claim 10 or 11, wherein the expression vector comprises the constitutive GAP promoter of P. pastoris. 14. The method for recombinant production according to claim 12, characterized in that the nucleic acid thus obtained is a plasmid preferably selected from the group pPLG11.2 pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 , and pPLG20.1, pAC37.1, pJW9.1, pMHS476.1, pSM54.2, pSM49.8, pSM82.1 and pSM58.1. 15. The method for recombinant production according to one or more of the previous claims, characterized in that the codons encode the cleavage site of the Kex2 protease and the plasminogen fusion gene has the nucleic acid sequence shown in FIG. I know that. ID No. 7, 13, 50, 52, 54, 56 or 58. 16. The method for recombinant production according to one or more of the previous claims, characterized in that the codons code for the cleavage site of the protease. Kex2 and the plasminogen fusion protein have the amino acid sequence shown in Seq. ID No. 8, 14, 40, 42, 44, 46 or 48.JW. 17. The method for recombinant production according to one or more of the previous claims, characterized in that the codons code for the cleavage site of the Kex2 protease and the Stel3 protease and the plasminogen fusion gene has the nucleic acid sequence shown in Seq. ID No. 9, 15, 51, 53, 55 or 57. 18. The method for recombinant production according to one or more of the previous claims, characterized in that the codons code for the cleavage site of the Kex2 protease and the Stel3 protease and the plasminogen fusion protein have the amino acid sequence shown in Seq. ID No. 10, 16, 41, 43, 45, 47, or 49. 19. Method for recombinant production according to one or more of the previous claims, wherein a host considered in the microorganisms is transformed with the included plasmid in claim 14. 20. The method for recombinant production according to one or more of the previous claims wherein the host organism used is a eukaryotic microorganism. 21. The method for recombinant production according to claim 20, characterized in that the host organism used is considered in the genus of fungus. 22. The method for recombinant production according to claim 21, characterized in that the host organism used is considered in the genus of Pichia, Saccharmyces, Hasenula, Candida or Aspergillus. 23. The method for recombinant production according to claim 22, characterized in that the host organism used is considered in the species Pichia pastoris, Pichia Methanolic, Hansenula polymorpha, Aspergillus niger, Aspergillus oryzae or Aspergillus nidulans. 24. The method for recombinant production according to one or more of the previous claims, wherein the nucleic acid sequence that is over-expressed codes for at least the functional part of the plasminogen and at least the functional part of the plasminogen that is secretory for a microbial host organism is transformed with the fusion product generated according to step a) 25. The method for recombinant production according to one or more of the previous claims, wherein the functional part of the acid sequence Plasminogen nucleic acids is one of the sequences Seq ID, No, 60, 61, 62, 63, 64, 65 or 66. 26. The method for recombinant production according to one or more of the previous claims, characterized in that produces a functional human plasminogen. 27. Method for recombinant reproduction according to one or more of the previous claims, characterized in that a plasminogen homologous to the functional human is produced. 28. Plasmid pPLG11.2, plasmid pPLG12.1, plasmid pPLG13.1, plasmid pPLG14.2, plasmid pPLG15.1, plasmid pPLG16.3, plasmid pPLG17.2, plasmid pPLG18.1, plasmid pPLG19.2, or plasmid pPLG20 .1. 29. Plasmid pMHS476.1, plasmid pSM54.2, plasmid pSM49.8, plasmid pSM82.1. 30. Plasmid pSM58.1, plasmid pAC37.1, plasmid pJW9.1. 31. The nucleic acid sequence obtainable by the method of recombinant production according to one or more of claims 1 to 27, characterized in that the nucleic acid sequence encoding at least the functional part of the plasminogen is operatively coupled a promoter that is activated in the yeast and additionally is coupled to a nucleic acid sequence coding for at least one signal peptide, the nucleic acid sequence encoding the functional plasminogen and the nucleic acid sequence encoding for at least the signal peptide with the codons for the cleavage sites of the Kex2 and Stel3 proteases. 32. The plasminogen obtainable through the recombinant production method according to one or more of claims 1 to 27. 33. The plasminogen according to claim 32, characterized in that it refers to a microplasminogen, miniplasminogen, Lys plasminogen, Glu plasminogen or a plasminogen derivative. 34. The plasminogen derivative according to claim 33, characterized in that it contains the proteolytic functional domain of plasminogen and comprises at least one deletion and / or at least one amino acid exchange and / or is fused with at least one amino acid or at least one peptide or at least one protein. 35. The plasminogen derivative according to claim 33 or 34, characterized in that it is activatable through at least one plasminogen activator to activate the plasmin. 36. The plasminogen derivative according to one of claims 33 to 35, characterized in that it contains at least the proteolytic functional domain of plasminogen and characterizes a sequence homology to micro-, mini-, Lys- or Glu-plasminogen of approximately 80%, preferably about 90% and especially preferred about 95%. 37. The plasmin obtainable through the activation of plasminogen or plasminogen derivatives according to one of claims 32-36 using at least one plasminogen activator. 38. The microbial host organism comprising the fusion product of a nucleic acid sequence derived therefrom obtained in step a) according to one of claims 1 to 27. 39. The microbial host organism according to claim 38 , characterized in that it is selected from the group Pichia pastoris, Pichia ethanolica, Saccharomyces cerevisiae, Hansenula polymorpha, Aspergillus niger, Aspergillus oryzae and Aspergillus nidulans. 40. The use of a functional plasminogen and / or the plasmin originating therefrom produced according to one of claims 1 to 27 for the preparation of a pharmaceutical. 41. The use of a functional plasminogen and / or the plasmin originating therefrom produced according to one of claims 1 to 27 for the treatment of wounds, thrombotic events or the prevention of thrombotic events. 42. The use of a functional plasminogen and / or the plasmin originating therefrom produced according to one of claims 1 to 27 as an anti-thrombotic agent as well as an active anti-coagulant 43. The use more particularly according to claim 42 for the prophylaxis and / or treatment of heart attack, stroke, thrombosis, venous thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, blood vessel inflammation, acute pulmonary embolism, acute and subacute arterial thrombosis, recent coagulations or past thrombosis venous, deep vein thrombosis of the hip and extremities, early thrombosis in the area of deobliterated vessels, acute occlusion of the central vessel in the eye, conjunctivitis in the case of type I deficiency of plasminogen, burn injuries, alkaline or acid burns and freezing, shock during disseminated intravasal coagulation, acute arterial occlusions of the extremities, arteriopathies or chronic clusters, thrombosis of arteriovenous shunts as well as for the treatment subsequent to the cardiac attack, subsequent to a revascularization by surgery, subsequent to an angioplasty as well as subsequent to a distention of the cavity, for the thrombotic therapy in the case of acute cardiac attack, for recanalization of arteriovenous shunts, as well as for reperfusion of occluded coronary arteries in the case of acute heart attack. 44. The use according to one or more of claims 40 to 43 in combination with an anticoagulant. 45. The use according to claim 44, characterized in that the anticoagulant refers to heparin, heparin derivatives, or acetylsalicylic acid. 46. The use of a functional plasminogen and / or the plasmin originating therefrom, produced according to one of claims 1 to 27 to be incorporated in restorative materials, plasters or for use in combination with cicatricial drugs. 47. The restoration materials bandages and cicatricial plasters comprising functional plasminogen and / or the plasmin originating from this produced according to one of claims 1 to 27. 48. The restoration materials, bandages and cicatricial plasters in accordance with claim 47, comprising 0.01-500 U of plasminogen and / or plasmin originating therefrom per cm 2 of the pharmaceutical formulation, preferably 0.1-250 Units and especially 1- 150 Units of plasminogen and / or a plasmin that originates from this per cm2 of the pharmaceutical formulation. 49. The use of restoration materials, bandages and cicatricial plasters according to claim 47 or 48 for the treatment of burn injuries, freezing, alkaline or acidic burns, injuries and / or injuries. 50. The pharmaceutical composition comprising a plasminogen and / or a plasmin originating from this, produced according to one or more of claims 1 to 27, and a pharmaceutically acceptable vehicle, additive and / or solvent, as well as in case or need of anticoagulation active agents. 51. The pharmaceutical composition according to claim 50 suitable for oral, topical, or parenteral, intravasal, especially intravenous, intraperitoneal, subcutaneous or intramuscular application. 52. The pharmaceutical composition according to claim 51 suitable for topical application containing 0.01-500 U of plasminogen and / or a plasmin originating therefrom, per gram of the pharmaceutical composition, preferably 0.1-250 Units and especially preferred is 1- 150 Units of plasminogen and / or a plasmin originating therefrom per gram of the pharmaceutical composition. 53. The pharmaceutical composition according to claim 51 suitable for oral application containing 0.01 - 100,000 U of plasminogen and / or a plasmin originating therefrom per gram of the pharmaceutical composition, preferably 100-80,000 Units and it prefers especially from 1,000-50,000 Units of plasminogen and / or one plasmin that originates from this per gram of the pharmaceutical composition. 54. The pharmaceutical composition according to claim 51 suitable for injection or infusion containing 0.1 - 100 million U of plasminogen and / or a plasmin originating from this per 10 ml of solution, preferably 1-10 million. Units and 3 to 5 million Units of plasminogen and / or a plasmin originating from it per 10 ml of the injection or infusion solution are especially preferred. 55. The use of the pharmaceutical composition according to one of claims 50 to 54 for the prophylaxis and / or treatment of heart attack, stroke, thrombosis, venous thrombosis, restenosis, hypoxia, ischemia, coagulation necrosis, vessel inflammation. blood, acute pulmonary embolism, acute and subacute arterial thrombosis, recent or past coagulation of venous thrombosis, deep vein thrombosis of the hip and extremities, early thrombosis in the area of de-blistered vessels, acute occlusion of the central vessel in the eye, conjunctivitis in the case of type I deficiency of plasminogen, burn injuries, alkaline or acidic burns and freezing, shock during disseminated intravasal coagulation, acute arterial occlusions of the extremities, chronic occlusive arteriopathies, thrombosis of arteriovenous shunts as well as for the treatment subsequent to the attack heart, subsequent to a revascularization by surgery, subse count for an angioplasty as well as subsequent distention of the cavity, for thrombotic therapy in the case of acute heart attack, for recanalization of arteriovenous shunts, as well as for reperfusion of occluded coronary arteries in the case of acute heart attack. The vector comprising the fusion product or a nucleic acid sequence derived therefrom obtained in step a) according to claim 1. 57. The DNA molecule comprising the fusion product or a nucleic acid sequence derived from the same obtained in step a) according to claim 1. 58. The RNA molecule comprising the fusion product or a nucleic acid sequence derived therefrom obtained in step a) according to claim 1 59. The selection method for identification of plasminogen activators through the use of functional plasminogen according to claim 36 or 37. 60. The method of selection according to claim 59, characterized in that the activity of the resulting plasmin is measured after preincubation of the proteases with the functional plasminogen according to claim 35 or 36. 61. The method of selection of according to claim 59 or characterized in that the activity of the resulting plasmin is measured with a synthetic peptide substrate. 62. The method of selection according to claim 61, characterized in that the activity of the resulting plasmin is measured with N-tosyl-Gly-Pro-Lys-pNA.